Heparin-like compounds, their preparation and use to prevent arterial thrombosis associated with vascular injury and interventions

ABSTRACT

Heparin-like compounds inhibit collagen-induced platelet aggregation in flowing whole blood. The compounds share properties displayed by native mast-cell derived heparin proteoglycans (HEP-PG) and/or heparin glycosaminoglycan (HEP-GAG) molecules. The compounds are useful in prevention and treatment of severe vascular disorders including arterial thrombosis.

PRIORITY

The present application is a continuation of U.S. Ser. No. 11/826,668,filed Jul. 17, 2007 now U.S. Pat. No. 7,504,113, which is a continuationof U.S. Ser. No. 10/418,095, filed Apr. 18, 2003 now U.S. Pat. No.7,314,860, which is a division of Ser. No. 09/230,097, filed Jan. 20,1999 now abandoned, which is a National Stage of PCT/FI98/00925, filedNov. 25, 1998, and claims priority to Finland Application No. 974321,filed Nov. 25, 1997, all of which are incorporated herein by reference.

THE TECHNICAL FIELD OF THE INVENTION

The invention is related to heparin-like compounds characterized bytheir capacity of almost complete inhibition of collagen-inducedplatelet aggregation in flowing whole blood and a coupling density ofnegatively charged heparin or heparin-like glycosaminoglycan units thatgives them the unique properties first displayed in native mastcell-derived heparin proteoglycans (HEP-PG) or heparin glycosaminoglycan(HEP-GAG) molecules obtainable thereof. The present invention is alsorelated to methods for preparing said heparin-like compounds and theiruse in prophylactic treatment of arterial thrombosis associated withvascular injuries and interventions.

THE BACKGROUND OF THE INVENTION

Heparin is a glycosaminoglycan, an acidic mucopolysaccharide composed ofD-glucuronic acid and D-glucosamine with a high degree of N-sulphation.It is present in the form of proteoglycan in many mammalian tissues,such as the intestine, liver, lung, being localized in the connectivetissue-type mast cells, which line for example the vascular and serosalsystem of mammals. The main pharmaceutical characteristic of heparin isits ability to enhance the activity of the natural anticoagulant,antithrombin III.

Heparins exist naturally bound to proteins, forming so called heparinproteoglycans. Usually, the endogenous or native, naturally existingheparin proteoglycans contain 10-15 heparin glycosaminoglycan chains,each chain having a molecular weight in the range of 75±25 kDa, andbeing bound to one core protein or polypeptide. Each native heparinglycosaminoglycan chain contains several separate heparin unitsconsequently placed end-to-end, which are cleaved by endoglycosidases intheir natural environment. The natural or native conjugates aredifficult to prepare in pure form. Thus, they have not been suggestedfor therapeutical or corresponding use. Heparin glycosaminoglycansbelong to a larger group of negatively charged heteropolysaccharides,which generally are associated with proteins forming so calledproteoglycans. Examples of other naturally existing glycosaminoglycansare for example chondroitin-4- and. 6-sulphates, keratan sulphates,dermatan sulphates, hyaluronic acid, heparan sulphates and heparins. Ofsaid heparin-like compounds existing in nature, only hyaluronic acid isgenerally not associated with a proteinaceous core molecule.

During the past decades the trend in heparin research has been todevelop and use heparin chain units, which have been fractionated forsystemic clinical preparations of shorter chains to increasespecificity. The generally used two types of standard clinical heparinsare the so called unfractionated or high-molecular weight heparins andfractionated or low-molecular-weight heparins. Said two types ofheparins have an average molecular weight of 15 and 5 kDa, respectively.In the present invention these two types of heparins are both consideredto be lower-molecular-weight heparins. Most commercial preparations havea molecular weight between 4 to 20 kDa depending on their origin, themethod of preparation and/or determination. Thus, the commercialheparins belong to the lower-molecular-weight heparins as defined in thepresent invention.

In the U.S. Pat. No. 5,529,986 a synthetic macromolecular heparinconjugate is described. It consists of at least 20 heparin moieties, butcan contain more than 100 heparin moieties, combined with natural orsynthetic substantially straight-chained polymer backbones such aspolylysine, polyornithine, chitosan, polyamine or polyallyl.

However, in said patent each heparin moiety is also characterized by alow molecular weight of approximately 12 kDa, which is far shorter thanthe heparin chains in native heparin proteoglycans. The macromolecularheparin molecule described in U.S. Pat. No. 5,529,986 is said to beespecially useful for coating surfaces in extracorporeal circulationsystems and its action is said to be based on binding to antithrombinIII and enhancement of its activity, which is the main functionalanticoagulant mechanism of all current heparin preparations.

Primarily, the standard heparin preparations are used for systemictreatment of thrombosis. As such they are most efficient inplatelet-poor thrombi, such as venous thrombi, where coagulationactivity prevails. The clinically used standard heparins, thougheffective in systemic treatment of thrombosis, by blocking the furthergrowth of thrombosis, are not effective enough to prevent thromboticcomplications, associated with either endogenous rupture of anatheromatous plaque or exogenous angioplasty or vascular ormicrovascular surgery.

Arterial interventions, such as angioplasty [PT(C)A=percutaneoustransluminal (coronary) angioplasty] with or without stenting andvascular or microvascular surgery as well as (directional) arterectomyand peripheral thrombendarterectomy, represent a growing modality oftreatment for cardiovascular diseases, which are the main cause ofdeath. Accordingly, platelet-driven arterial thrombosis, which occurs inconnection with endogenous vascular or microvascular injuries and/orexogenous interventions is a frequently encountered problem and in thesesituations the traditional systemic treatment of thrombosis is often oflimited efficacy.

Current systemic antithrombotic treatment in connection with arterialinterventions include the combination of an anticoagulant, such asunfractionated heparin (12 kDa) or low-molecular-weight heparins (7.5kDa) with an antiplatelet drug, such as acetylsalicylic acid(cyclooxygenase inhibitor), or ticlopidine or better clopidogrel (ADPantagonist). The latest development is represented by potent plateletglycoprotein IIb/IIIa, von Willebrand factor and fibrinogen receptorantagonists, such as abciximab, tirofiban and velofibatide. The newcombination treatments have succeeded in preventing 30-35% of acutethrombotic closures of the interventionally treated thrombus-pronevessels. The bleeding risk (major bleeding) requiring infusion of bloodproducts is around 6-7%. So far abciximab has had the best efficacy, butsince it is an antibody-based drug repetitive ad ministration may causeantigenicity.

The systemic treatment with unfractionated heparin suffers from manyunwanted effects, such as unpredictable bioavailability, shorthalf-life, unspecific binding to proteins leading to compromisedantithrombin III function and immunologic platelet effects withthrombocytopenia and thrombosis as well. These unwanted effects havebeen largely bypassed with the use of the low-molecular-weight,fractionated heparins, which however, have a limited capacity ininhibiting arterial thrombosis due to a limited control of fibrin-boundthrombin, and of platelet-bound factor Xa, and due to the neutralizationof heparin-activity by platelet-secreted platelet factor 4. Thus, thereis a great need for developing an effective and reliable prophylactictreatment of arterial thrombosis associated with vascular ormicrovascular injuries and interventions.

In their studies, the present inventors found that in contrast to thelower-molecular-weight heparins, native heparin proteoglycans (HEP-PG)obtainable from mammalian mast cells express potent antithromboticproperties, which are based on their capacity, to inhibitplatelet-collagen interactions. This unique property, namely blockingthe platelet activation events subsequent to platelet adhesion tocollagen, which is not present in the lower-molecular-weight, includingunfractionated and fractionated commercial heparins, occurssimultaneously with a potential to enhance the function or activity ofantithrombin III or heparin cofactor II.

In contrast to the traditional heparin mechanism, i.e. the antithrombinIII enhancing action of heparins in the current clinical use as well asthat of the macromolecular heparin construct described in U.S. Pat. No.5,529,986 the efficacy of the heparin-like compounds of the presentinvention does not depend on the antithrombin III activity, but is basedon a previously undescribed mechanism of strong inhibition of plateletactivation triggered by platelet adhesion upon collagen. The exactmechanism is presently unknown, but is supposed to depend on disruptionof the activation signal following platelet GP Ia/IIa binding tocollagen and subsequent membrane phospholipid flip-flop and procoagulantactivity, normally induced by collagen in platelets. Also, strongbinding to von Willebrand factor (vWF) may be involved.

The present invention provides an alternative for antiplatelet treatmentin form of local application, which can be combined with a systemicantiplatelet drug. This combination can be used in conjunction withangioplasty, vascular and microvascular surgery and endarterectomy topassivate the exposed subendotheliai vascular and microvascular collagenfor adhering platelets. During the initial studies, the desired effectachieved with the heparin proteoglycans (HEP-PG), was a significantlyreduced local thrombus formation based on inhibition ofplatelet-to-platelet interaction, but preserved adhesion upon collagen.The collagen-induced platelet activation upon the adherent platelets wasshown to be fully blocked in the presence of mast cell-derivable heparinproteoglycans (HEP-PG), multiple heparin glycosaminoglycan molecules, assuch or connected to core molecules and lower-molecular-weight heparinor heparin-like glycosaminoglycans connected to spheroidal or globularcore molecules to provide the spatially optimal macromolecularpresentation or configuration which provides a sufficient couplingdensity of negatively charged glycosaminoglycans.

The above defined effect was obtainable both when the heparinglycosaminoglycan. (HEP-GAG) having a molecular weight of 75±25 kDaand/or multiple carrier—(core molecule) coupled, spacer/linker-provided,unfractionated or fractionated heparin or heparin-like chains presentedin the optimal spatial configuration and proteoglycan (HEP-PG) moleculescomprising said HEP-GAG-molecules were in solution or immobilized oncollagen-coated surfaces or administrated to vessel surfaces to beclosed as anastomosis. Thrombin targeting with the current means toprevent thrombosis and to limit excessive wound repair, has still beenassociated with development of restenosis at the treated site. Theadvantage of the HEP-GAG- and HEP-PG-molecules of the present inventionwas preserved systemic platelet function which ensured normal hemostaticresponses. The local inhibitory effect of mast cell-derived HEP-GAG- andHEP-PG-molecules on smooth muscle cell proliferation in vitro was alsoshown to be significantly better than that of lower-molecular-weightheparin species (Wang & Kovanen, Circulation Res, In Press).

Based on these preliminary findings the present inventors developed theheparin-like compounds of the present invention, as well as methods fortheir preparation and their use. The objectives of the invention are setforth below.

The first objective of the present invention was to provide solubleheparin-like compounds, mimicking the structure and properties of themast cell-derived multiple heparin glycosaminoglycans (HEP-GAG) and/orheparin proteoglycans (HEP-PG), which HEP-GAG- and HEP-PG-molecules hadbeen shown to be characterized by a hitherto unreported mechanism basedon an almost complete inhibition of platelet-collagen interaction. Saidmechanism is useful and convenient for screening and determining theproperties of newly developed, synthetically, semisynthetically orbiotechnologically modified heparin-like compounds as well as of locallyapplicable preparations useful for preventing thrombosis associated withvascular or microvascular injuries and interventions, such asangioplasty, stenting and vascular grafting.

Another objective of the invention was to provide pharmaceuticallyuseful preparations, which comprise the heparin-like compounds of thepresent invention in combination with compatible adjuvants, carriers,etc.

A third objective of the invention was to provide means for and/ordevices for administering the heparin-like compounds of the presentinvention by coating said means or devices.

The objective of the invention was also to provide methods for preparingsaid HEP-GAG- and HEP-PG-molecules from mast cells and to further modifythe mast cells or the HEP-GAG- or HEP-PG-molecules by′ chemical and/orbiotechnological methods to provide novel heparin-like compounds, withoptimal spatial configurations and properties which are defined in theclaims and which are similar to those of the native mast cell-derivableHEP-PG-molecules or the multiple HEP-GAG-molecules obtainable therefrom.

Still a further objective of the present invention was the use of theheparin-like compounds of the present invention as such or asingredients for manufacturing preparations and devices useful forprophylactic treatment and prevention of severe vascular disordersincluding arterial thrombosis in connection with vascular andmicrovascular surgery or interventions, such as angioplasty, stentingand vascular grafting.

THE SUMMARY OF THE INVENTION

Platelet-collagen interactions are the essential triggering event inhemostasis and developing arterial thrombosis. In their preliminarystudies, the inventors found that there is a clear connection betweenthe molecular weight of the heparin .proteoglycans (HEP-PG)—especiallythe high molecular weight of the heparin proteoglycans (HEP-PG) based onthe multiple structure or the spatial configuration or presentation ofits heparin glycosaminoglycan (HEP-GAG) moieties—and their inhibitoryeffects on platelet-collagen interaction. It was shown that the bestresults were obtainable with multiple glycosaminoglycans (HEP-GAG) orheparin proteoglycans (HEP-PG) containing multiple HEP-GAG chains havinga size, which mimics the situation in vivo, wherein vascular mast cellswere activated and excreted their granules into the external bodyfluids, wherein the granulate-derived heparin molecules solubilized.Said solubilized heparin proteoglycans (HEP-PG) contained in averageabout 10 heparin glycosaminoglycan (HEP-GAG) moieties, each with amolecular weight of 75±25 kDa. The desired effect was also obtained bycombining several unfractionated or fractionated, herein so calledlower-molecular-weight heparin glycosaminoglycan units end-to-end orend-to-side to form multiple glycosaminoglycans, either as such orconnected to core molecules. Also similar inhibition of aggregation wasfound, when multiple amino-sulphated groups of multiple unfractionatedheparin chains (12±10 kDa) were coupled with a heterobifunctionalcoupling reagent, i.e. a spacer or linker molecule, such asN-succinylimidyl-3(2-pyridylthio)propionate (SPSD) to lysine residuespresent in albumin, a globular protein, offering an optimal coremolecules for producing the optimally charged heparin-like compounds ofthe present invention with the spatial configuration and couplingdensity.

Thus, the present invention is related to heparin-like compounds, whichcomprise multiple heparin or heparin-like glycosaminoglycan molecules,which have a high molecular weight and consist of several end-to-endand/or end-to-side connected heparin or heparin-like glycosaminoglycanmolecules as such or connected to a natural or synthetic, chain-like,preferably short or globular core molecule or lower-molecular-weightheparin or heparin-like glycosaminoglycans conjugated to a globular coremolecule. Preferably, spacer or linker molecules, which allow attachmentof more heparin or heparin-like molecules than the core moleculesthemselves are used to provide, the desired, sufficient coupling densityof said heparin or heparin-like glycosaminoglycan molecules. Theheparin-like compounds of the present invention, advantageously, have acoupling density of negatively charged heparin or heparin-likeglycosaminoglycan molecules or units, which provides the heparin-likecompounds of the present invention with a spatial configuration, whichseems to be responsible for or closely related to the unique andspecific properties of the heparin-like compounds of the presentinvention and which properties were first recognized with the mastcell-derived HEP-GAG- and HEP-PG-molecules, said property being thecapacity of substantially complete inhibition of the plateletaggregation upon collagen in flowing blood, which is a general cause ofarterial thrombosis associated with vascular or microvascular injury andinterventions.

The heparin-like compounds of the present invention preferably compriseseveral multiple end-to-end and/or end-to-side connected heparinglycosaminoglycan (HEP-GAG) molecules. Each of which should preferablyhave a molecular weight of 75±25 kDa or more than 75±25 kDa.

In the heparin-like compounds of the present invention the multipleheparin or heparin-like glycosaminoglycan molecules can be connected,coupled or conjugated to a natural or synthetic core molecule, whichpreferably is globular or provides the desired spheroidal configuration,but they can also be connected˜ to more chain-like core molecules.

Also lower-molecular-weight heparin or heparin-like glycosaminoglycanmolecules can be connected to core molecules. However, in such casesthe′ core molecule should have a spheroidal or globular configuration.It is also recommendable to use spacer or linker molecules, which allowcoupling of much more heparin or heparin-like molecules or units andthus provides a more optimal spatial configuration and a higher couplingdensity.

The core molecules are advantageously proteins or polypeptides. Usefulexamples of core molecules are globular proteins, such as albumin,preferably serum albumin of human origin. Another type of core moleculesis a polypeptide, which need not be very long and comprises e.g. one ormore repetitions of the Ser-Gly-Ser-Gly-sequence. Alternatively, otherkinds of amino acid sequences can be used.

The present invention describes methods for producing the heparin-likecompound both from natural sources, synthetically or semisyntheticallyor by biotechnological methods, including genetical modifications fromcommercially available heparins and proteins or polypeptides.

Natural heparin-like compounds are prepared by allowing isolated andpurified connective tissue-derived mast cells to grow in a suitable cellculture medium using conditions allowing good cell proliferation andproduction of heparin-containing granules. After the growth step hasbeen completed, i.e. when the yields of the heparin proteoglycans areoptimal, the heparin proteoglycan-containing granules are released byoptional activation and/or lysis. The activation step can be carried outwith mast cell agonists, which induce mast cell degranulation andrelease solubilized, multiple HEP-PG-molecules. Preferred agonists areselected from a group consisting of basic polyamines and calciumionophores. The released granules are allowed to solubilize in thesurrounding culture medium and said solubilized heparin proteoglycan(HEP-.PG) is collected from the exterior medium. Thereafter, if desired,native multiple heparin glycosaminoglycans (HEP-GAG) can be separatedfrom said heparin-proteoglycans and said heparin glycosaminoglycan(HEP-GAG) molecules can be further coupled to each others in order toobtain heparin glycosaminoglycans with a higher degree of branchingand/or multiplicity.

In the synthetic or semisynthetic methods several heparin orheparin-like glycosaminoglycan units can be connected end-to-end and/orend-to-side by covalent bonds. Optionally, especially when usinglower-molecular-weight heparin molecules as starting material, couplingreagents, such as spacer or linker molecules, should be used to providethe optional multiplicity and spatial configuration.

The present invention is above all related to a method for prophylactictreatment of arterial thrombosis associated with vascular ormicrovascular injuries and/or interventions. The method is performed bylocal administration of the heparin-like compounds of the presentinvention. The local administration is performed by directly applying aneffective amount of the heparin-like compounds of the present inventionin the form of preparations and/or indirectly as devices, such asstents, vascular grafts or extracorporeal circulation systems coatedwith the heparin-like compounds of the present invention. Theheparin-like compounds of the present invention can be combined withpharmaceutically acceptable carriers and/or adjuvants to provide moreadvantageous preparations for application and administration.

The present invention also describes preparations, means and/or devicesfor local administration of the heparin-like compounds of the presentinvention in connection with vascular or microvascular injuries orinterventions. Effective local administration is obtained for example bymeans and/or devices coated with heparin-like compounds of the presentinvention.

The invention is also related to a method for preventing plateletaggregation or clogging at the site of vascular injury or intervention.The prevention is obtainable especially by the aid of devices coatedwith the heparin-like compounds of the present invention.

Methods for manufacturing means and/or devices for local administrationare also suggested and include for example contacting the devices with asolution containing the heparin-like compounds of the present inventionand treating the devices appropriately under sterile conditions, so thatthe activity is retained and storing is possible. The devices can forexample be dried, e.g. by lyophilization and stored in sterile vialsuntil used. Other ways of treating and storing the devices coated withthe heparin-like compounds of the present invention are known to thoseskilled in the art.

The present invention is thus related to the use of heparin-likecompounds for manufacturing medicines and/or devices capable ofsubstantially complete inhibition of platelet aggregation upon collagenin flowing whole blood, the cause of arterial thrombosis associated withvascular or microvascular injury and interventions.

THE SHORT DESCRIPTION OF THE FIGURES

FIG. 1 shows the dose-dependent effects of the various heparinglycosaminoglycans HEP-GAG on thrombin time in citrated plasma (dilution1:3). HMWH=high-molecular-weight or unfractionated heparin,LMWH=low-molecular-weight, or fractionated heparin, HEP-PG=mastcell-derived water soluble heparin proteoglycans.

FIG. 2 shows comparative effects of mast cell-derived HEP-PG (MW 750kDa), the heparin glycosaminoglycan (HEP-GAG) chains derived from HEP-PG(MW 75 kDa), HMWH (MW 15 kDa), and LMWH (MW 5. kDa) on collagen—(Sigma,collagen of the aggregation kit) induced platelet aggregation (25 pg/ml)maximal aggregation (%) in platelet rich plasma (PRP) anticoagulatedwith citrate.

FIG. 3 shows the effect of mast cell-derived HEP-PG (3 μg/ml) onplatelet deposition on collagen (bovine, type I, fibrillar) underdifferent shear rate conditions in PPACK-anticoagulant flowing wholeblood. Perfusion time was 5 minutes. Values are means±SD for four donorsat 200, 700 and 1700 1/s, all in duplicate.

FIG. 4 shows scanning electron micrographs of perfusion channels withimmobilized standard heparin (10 mg/ml) or mast cell-derived HEP-PG (10μg/ml) after human whole blood perfusions over monomeric collagen type Iunder the shear rate of 1640 l/s. In the top panel, control, largeplatelet aggregates can be detected in the perfusion channel. In themiddle panel standard unfractionated heparin (HMWH) somewhat reduced thesize of aggregates, but did not seemingly affect surface coverage. Inthe lower panel, in the presence of mast cell-derived HEP-PG, plateletdeposition was almost absent, only occasional adherent single platelets,mainly in the channel edges having the low shear rate conditions, weredetected.

FIG. 5 shows the effect of immobilized unfractionated standard heparin(10 mg/ml) and HEP-PG (10 μg/ml) on platelet deposition over collagentype I in fibrillar form (equine, Horm™ collagen Nycomed). Plateletdeposition to this form of collagen is dependent on GP IV and GP VI inaddition to GP Ia/IIa. Donors are five, all duplicate. Shear rate was1640 l/s, perfusion time was 5 min and blood was anticoagulated withPPACK 30 μM. Colt is collagen and HMWH is unfractionated standardheparin and HEP-PG is mast cell-derived heparin proteoglycan.

FIG. 6 shows the effect of immobilized unfractionated standard heparin(10 mg/mi) and mast cell-derived HEP-PG (10 μg/mi) on plateletdeposition over collagen type I in monomeric form, native aceticacid-soluble bovine collagen fibers digested with pepsin. Plateletdeposition is highly dependent on GP Ia/IIa. Both unfractionated heparinand HEP-PG inhibited platelet deposition when immobilized or monomericcollagen, however, HEP-PG was significantly more potent in itsinhibitory function. Donors are five, all duplicate. Shear rate was 1640l/s. perfusion time was 5 min and blood was anticoagulated with PPACK 30μM. Coil is collagen and HMWH is unfractionated, standard heparin andHEP-PG is mast cell-derived heparin proteoglycan.

FIG. 7 shows the effect of mast cell-derived HEP-PG on plateletinteraction with fibrillar type I collagen in Mg2+ (2 mM)-containingbuffer. The experiment comprised 100×10⁶/ml platelets at 22° C. understatic conditions leading to equal platelet adhesion. Data are means±SDof values for four donors, all in duplicate. PD is platelet deposition.

FIG. 8 shows the effect of mast cell-derived HEP-PG on plateletinteraction with collagen in Mg2+ (2 mM)-containing buffer. Theexperiment comprised 300×10⁶/ml platelets at 37° C. under slow rotation(100 rpm) permitting platelet interaction on fibrillar type Icollagen-adherent platelets. Data are means±SD of values for fivedonors, all in duplicate. *p<0.000l, paired t-test. PD is plateletdeposition.

FIG. 9 shows scanning electron micrographs of the rat anastomosis modelafter 10 minutes circulation of blood over the vessel. Twomagnifications, ×75 and ×500 of the perfused anastomosis area. Theleft-sided panels represent saline-treated anastomosis and theright-sided panels represent mast cell-derived HEP-PG-treatedanastomosis. These experiments are representative of 5 differentexperiments.

FIG. 10A shows the collagen-induced (Sigma, aggregation kit, at 25μg/ml) platelet aggregation in citrated PRP.

FIG. 10B shows the effect of mast cell-derived HEP-PG (at 3 μg/ml ofheparin) on collagen-induced platelet aggregation.

FIG. 10C shows the effect of albumin-coupled unfractionated heparin (at0.75 μg/mi of heparin) on collagen-induced platelet aggregation).

THE DETAILED DESCRIPTION OF THE INVENTION Definitions

In the present description the following abbreviation and acronyms arefrequently used. GP means glycoprotein. HEP-GAG means long (75 kD) orbranched or multiple heparin glycosaminoglycans, which are capable ofalmost complete inhibition of collagen-induced platelet aggregation inflowing whole blood. HEP-PG means heparin proteoglycans (natural orsynthetic or semisynthetic and core molecule-coupled, i.e. protein- orpeptide-coupled) comprising the above defined HEP-GAG molecules. HMWHmeans commercially available, unfractionated high-molecular-weight,7.5-30 kDa heparins or heparin glycosaminoglycan units. LMWH meanscommercially available, low-molecular-weight, less than 7.5 kDa heparinsor heparin glycosaminoglycan units. Said two types of heparins are inthe present invention so called lower-molecular-weight heparin moleculesor units. HSA means human serum albumin, BSA means bovine serum albuminand MW molecular weight. PBS means phosphate-buffered saline, PPACKD-phenylalanyl-1-propyl-1-arginine chloromethyl ketone, PRPplatelet-rich plasma, SDS sodium dodecyl sulfate, TESN-Tris(hydroxy-methyl)methyl-2-aminoethane sulfonic acid and vWf vonWillebrand factor. SPDP meansN-succinylimidyl-3-(2-pyridylthio)propionate.

In the present description the terms used have the same meaning as theygenerally have in medical sciences, including immunochemistry,immunology, biochemistry, etc., as set forth in textbooks and reviewarticles in said fields. Some terms are, however, used more extensivelyand have a meaning that somewhat differs from the general use of theterm. Some of these terms are defined below.

The term “heparin-like compound” means compounds which have a structureresembling that of mast cell-derived heparin proteoglycans and heparinglycosaminoglycans and which are characterized by their capacity ofalmost complete inhibition of collagen-induced platelet aggregation inflowing whole blood and a coupling density of negatively charged heparinor heparin-like glycosaminoglycan units that gives them the uniqueproperties displayed by the native mast cell-derived heparinproteoglycans (HEP-PG) or heparin glycosaminoglycan (HEP-GAG) moleculesobtainable thereof and which property can be measured by the method(s)described in Lassila R, Lindstedt K, Kovanen P T. Arteriosclerosis,Thrombosis, and Vascular Biology 1997; 17 (12):3578-3587.

The heparin-like compounds of the present invention, either soluble orimmobilized on collagen, are above all characterized by their capacityfor inhibiting the platelet aggregation upon collagen in flowing wholeblood, which is a cause of arterial thrombosis associated with vascularinjury and interventions as well as their capacity for preventing theinteraction of flowing whole blood with collagen. Said properties, inaddition to the multiple structure or the optimal coupling density ofthe heparin or heparin-like glycosaminoglycan molecules, are theprerequisites required of the heparin-like compounds of the presentinvention.

In its most specific meaning the term “heparin-like compounds” islimited to mast cell-derived heparin proteoglycans (HEP-PG) and heparinglycosaminoglycans (HEP-GAG) obtainable thereof. However, it alsoincludes multiple, unfractionated or fractionated heparin orheparin-like chains coupled to core molecules, either directly or by aidof spacer or linker molecules to provide heparin-like compounds with aunique, spatially optimal configuration, which provides the compoundswith a desired, high coupling density of negatively charged heparin orheparin-like glycosaminoglycan molecules or units. The desired highcoupling density which provides the unique properties of theheparin-tike compounds of the present invention was first found forexample in native mast cell-derived heparin proteoglycans (HEP-PG) orthe multiple heparin glycosaminoglycan (HEP-GAG) molecules and it seemsto explain their properties as well.

The term “heparin or heparin-like proteoglycan” in the present inventionmeans such heparin or heparin-like proteoglycans, which fulfill theprerequisites set up in the definition of heparin-like compounds.Preferably the proteoglycans contain more than three multiple heparin orheparin-like glycosaminoglycan molecules bound to a core molecule. Theterm “heparin or heparin-like compounds” above all covers native,water-soluble heparin proteoglycans (HEP-PG) obtainable from mammalianconnective tissue type mast cells, either by tissue extraction orpreferably by cell cultivation. These heparin proteoglycans (HEP-PG)usually comprise approximately 10-15 multiple heparin glycosaminoglycan(HEP-GAG) molecules. Synthetically produced heparin or heparin-likeproteoglycans can comprise any number of multiple heparin orheparin-like. glycosaminoglycan molecules as long as they fulfill theabove defined prerequisites.

The term “water-soluble or solubilized heparin-like compounds” meansthat the mast cell-derived HEP-PG-molecules are released from thegranules of the mast cells into the exterior medium in which theysolubilize and thereafter can be separated from cell debris and othernon-soluble components. It is to be observed that the other heparin-likecompounds of the present invention should be provided with the same typeof solubility as defined above, even if it is an inherent property ofall said molecules to easily adhere to certain, especially solidsurfaces.

The term “multiple heparin or heparin-like glycosaminoglycan molecule”above all includes the native heparin glycosaminoglycan (HEP-GAG)molecules obtainable from the mast cell-derived heparin proteoglycansand having a molecular weight of 75±25 kDa. The term also includessynthetically or semisynthetically obtainable heparin or heparin-likeglycosaminoglycans with several end-to-end and/or end-to-side coupledglycosaminoglycan units, obtainable for example from unfractionated orfractionated lower-molecular-weight heparins. Said “multiple heparin orheparin-like glycosaminoglycan molecules” form multiple straight orbranched molecules with the properties set forth in the definition ofheparin-like compounds. Each heparin or heparin-like glycosaminoglycanmolecule should have a sufficient molecular weight to provide theproperties set out in the definition of the heparin-like compounds.

The “multiple heparin or heparin-like glycosaminoglycan molecules” orunfractionated or fractionated heparin or heparin-like chains can beconjugated to natural, synthetic or semisynthetic core molecules, forexample proteins, preferably globular proteins to provide heparinproteoglycans with the properties defined above.

The term “multiple heparin or heparin-like glycosaminoglycan molecules”include molecules having for example at least 3-4 end-to-end orend-to-side connected heparin or heparin-like glycosaminoglycan units,each having a molecular weight corresponding to the commerciallyavailable unfractionated or fractionated lower-molecular-weightheparins. Their molecular weight is generally less than 20 kDa, mostlyless than 15 kDa but also heparin glycosaminoglycans with molecularweight of less than 12 kDa can be used. In such case, only more of themhave to be combined in order to give the HEP-GAG-molecules which have asufficiently high MW or coupling density to provide the propertiesdefined in connection with the heparin-like compounds. Native mastcell-derived naturally multiple heparin glycosaminoglycan (HEP-GAG)molecules generally have a molecular weight of about 75±25 kDa. Suchnatively “multiple heparin glycosaminoglycan molecules” can also becoupled end-to-end and/or end-to-side in order to give larger molecules,which also belong to the scope of the present invention. Thus, themultiple heparin glycosaminoglycan (HEP-GAG) molecules of the presentinvention have a molecular weight of at least 75±25 kDa, including anymultiples thereof. Heparin-like glycosaminoglycans do not necessarilyhave to have the defined molecular weight, if they otherwise have thecharacteristic features of heparin-like compounds as defined in theclaims. In addition, to the high molecular weight, an alternative way ofproviding sufficient coupling density is provided by couplingunfractionated or fractionated glycosaminoglycans, e.g. heparins toglobular core molecules. The desired density′ is especially obtainableby using specific spacer or linker molecules, which allow′ the bindingof much more of the desired heparin or heparin glycosaminoglycan units.

Even if the native mast cell-derived HEP-GAG- and HEP-PG-molecules werecharacterized by having in plasma substantially low antithrombin-bindingactivities, this is not true for all the heparin-like compounds of thepresent invention. However, the capability of substantially completeinhibition of collagen-induced platelet aggregation, which is a cause ofarterial thrombosis associated with vascular or microvascular injury andinterventions should be present in all heparin-like compounds of thepresent invention, but in some of them said property is combined withpotent antithrombin III accelerating activities. Said combined effectgives an additional advantageous property to the product, i.e. to thecompounds, preparations and devices of the present invention.

Thus, the terms “heparin or heparin-like glycosaminoglycan” and “heparinor heparin-like proteoglycan” in the present invention means not onlymast cell-derivable heparin proteoglycans (HEP-PG) or heparinglycosaminoglycans (HEP-GAG), but also include heparin-likeglycosaminoglycans and heparin-like proteoglycans, which are producedsynthetically, semisynthetically or using recombinant DNA techniques. Inits broadest aspect the term “heparin-like compounds” means linear orbranched heparin-like glycosaminoglycans, i.e. compounds composed ofhundreds of monosaccharides comprising amino-groups and being connectedor covalently attached to natural or synthetic or semisynthetic coremolecules. Such heparin-like compounds are obtainable from naturallyoccurring glycosaminoglycan species such as chondroitin sulphates,keratan sulphates, dermatan sulphates, heparan sulphates and/orhyaluronic acid, either as such or modified by chemical orbiotechnological means, including recombinant-DNA-techniques to providemolecules, which fulfill the requirements and prerequisites set outabove and which are characteristic features of the native mastcell-derivable heparin proteoglycans (HEP-PG) and heparinglycosaminoglycans (HEP-GAG). It is not necessary to isolate theheparin-like glycosaminoglycan molecules from nature. They can be alsosynthesized or fragments of naturally occurring species can be coupledtogether especially with heparin-fragments to provide new variants, theproperties of which can easily be tested by known methods by thoseskilled in the art.

The term “mast cell” means mast cells related to connective tissue, suchas vascular or serosal mast cells of mammalian or human origin, whichcan be isolated from respective tissues and which cells can becultivated in a culture medium under conditions, which allowproliferation of the cells and secretion of the heparin proteoglycans(HEP-PG) of the present invention upon lysis and/or activation of the−mast cells. Mammalian mast cells, including human mast cells, areavailable and described in literature (Butterfield J H, Weiler D, DewaldG, Gleich G J, Leuk Res 1 988; 12: 345-55; Nilsson G, Blom T,Kusche-Gullberg M, Kjellen L, Butterfield J H, Sundstom G, Nilsson K,Hellman L. Scand J Immunol 1994; 39: 489-98). The mast cells can begenetically modified, by conventional mutagenesis or recombinant DNAtechniques.

The term “coupling density” means that negatively charged heparin orheparin-like glycosaminoglycans can be spatially brought together insuch a way that the characteristic features of the heparin-likecompounds of the present invention are obtainable. This is achievedeither by preparing multiplied heparin or heparin-likeglycosaminoglycans or by bringing shorter molecules together in aspatial configuration, which allows a sufficient concentration andoptimal presentation of negative charges.

The term “spacer/linker molecules” means polymeric compounds providedwith a multitude of groups which allow binding of the desired compoundsor molecules to the core molecule. SPSD is one example, but thoseskilled in e.g. protein chemistry and combinatorial chemistry, can finda multitude of other equally advantageous spacer/linker molecules.

The term “capacity of substantially complete inhibition of plateletaggregation on collagen in flowing whole blood” means that theinteraction between platelets and collagen can be prevented as measuredby the methods set forth in Lassila R, Lindstedt K, Kovanen P T.Arteriosclerosis, Thrombosis, and Vascular Biology 1997; 17 (12):3578-3587.

The term “local administration” or “local use” means that theheparin-like compounds of the present invention are administered to theinjured region of the blood vessel as such or in the form of in situ oron place, i.e. locally or topically applicable preparations orcompositions and/or as means or devices. The local administration isperformed for example by flushing the exposed vascular tissue with thesolution. The heparin-like compounds of the present invention can beused for coating devices, which are placed in contact with the injuredblood vessels. Such devices are for example “coated stents” i.e. smalldevices, which are placed into the blood vessels to keep them open or“vascular grafts” which are used to replace weakened vascular tissues.Other examples of devices, which can be coated are for exampleextracorporeal circulation systems, the inner walls of which can becoated with the heparin-like compounds of the present invention.

The term “prophylactic treatment” means prevention of thrombosis inassociation with vascular or microvascular injuries and interventions bylocal or topical application, e.g. by flushing of the exposed vasculartissue with the heparin-like compounds of the present invention as suchor as suitable preparations or in form of devices coated with saidheparin-like compounds. The local administration of the heparin-likecompounds can be used as such or in combination with systemicadministration of conventional preparations.

The term “arterial thrombosis” means thrombosis in arteries inassociation with endogenous conditions, including atherotrombosis, andother hemostatic conditions in vasculature, vascular injuries andiatrogenic angioplasty and other interventions.

The term “preparation” means the heparin-like compounds of the presentinvention used in combination with compatible, pharmaceuticallyacceptable adjuvants, carriers in preparations, means and/or devices foradministration to the patient.

THE GENERAL DESCRIPTION OF THE INVENTION

The primary aim of the present invention was to compare the effects ofthe HEP-GAG- or HEP-PG-molecules of the present invention and standardheparins (average MWs of 15 and 5 kDa) on platelet-collagen interactionin vitro. In contrast to standard heparins, mast cell-derived HEP-PG-and HEP-GAG-molecules were shown to completely inhibit collagen-inducedplatelet aggregation and serotonin release in platelet-rich plasma. Theinhibition caused by mast cell-derived HEP-GAG- and/or HEP-PG-moleculesof the present invention was shown to be dependent on theirmacromolecular structure. In flowing blood, mast cell-derived HEP-GAG-and HEP-PG-molecules also inhibited platelet deposition on acollagen-coated surface both at low and high shear rates. Although, themast cell-derived HEP-GAG- and HEP-PG-molecules did not blockglycoprotein (GP) Ia/IIa-mediated platelet adhesion, they attenuatedsubsequent platelet activation and aggregation, as well as fibrinogenbinding to platelets after collagen stimulation. The mast cell-derivedHEP-GAG- and HEP-PG-molecules did not bind to platelets, but weretightly bound to von Willebrand Factor (vWf) enhancing its binding tocollagen. While platelet adhesion at high shear rate and vWf binding toGP Ib after ristocetin stimulation was not markedly affected, theHEP-GAG- and HEP-PG-molecules of the present invention reducedthrombin-induced aggregation and vWf binding to GP IIb/IIIa. Thesefindings implied that activation of vascular mast cells with ensuingsecretion of HEP-PG may locally attenuate the thrombogenicity of matrixcollagen by inhibiting its platelet-activating capacity.

To study the ability of the mast cell-derived HEP-PG to inhibit thrombinactivity was the first objective of the study. Particularly, theinhibitory effect of the very high-molecular-weight heparin species wascompared with the well-known anticoagulative function of standardheparins, which essentially relies on the potentiation of antithrombinIII activity (Nader H B, Dietrich C P., in Lane D A and Lindahl U (eds):Heparin: Chemical and Biological Properties, Clinical Application.Erward Arnold, London. 1989, pp 81-96). The assays focusing on thedifferential capacities of HEP-PG, HMWH, and LMWH to inhibit thrombin(FIG. 1) showed that, in the absence of plasma proteins, HEP-PG had abetter functional ability than HMWH to potentiate antithrombin IIIdirectly, whereas in the presence of plasma, HEP-PG were less potentthan HMWH. These data could be understood if HEP-PG was, moreeffectively than HMWH, bound to plasma proteins, such as vWf, which werebelieved to compete with antithrombin III for binding to heparin. It hasbeen shown that the greater the molecular weight of the heparin, thehigher is its affinity for plasma proteins (Baruch D, Ajzenberg N, DenisC, Legendre J-C, Lormeau J-C, Meyer D. Thromb Haemost 1994; 71: 141-146;Young E, Prins M, Levine M N, Hirsh J. Thromb Haemost 1992; 67:639-543). In all, it was concluded that the inhibitory potential ofHEP-PG upon the observed platelet-collagen interaction (see Examples15-16 below), as compared with HMWH and LMWH, could not be due toantithrombin III-dependent thrombin inhibition.

The major findings of the present study were the total inhibition byHEP-PG of (i) collagen-induced platelet aggregation (FIG. 2), (ii)subsequent dense granule release (serotonin), and (iii) plateletdeposition on immobilized collagen in flowing whole blood under both lowand high shear rate conditions (FIG. 3). Previously, HMWH. (atconcentrations >6 μg/ml) had been shown to impair platelet aggregationinduced with a low-dose collagen in cation-depleted PRP (Fernandez F,N'guyen P. van Ryn J, Ofosu F A, Hirsh J, Buchanan M R. Thromb Res.1986; 43: 491-495). In the present studies, it was found that mastcell-derived HEP-PG, in contrast to HMWH, totally inhibitedcollagen-induced platelet aggregation and serotonin release,irrespective of collagen concentration. Curiously, HEP-PG-molecules wereshown to be able to prevent the action of collagen on platelets evenwhen added to the platelet-rich plasma (PRP) 10 s after collagen. Mastcell-derived HEP-PG-molecules were shown to inhibit platelet aggregationby collagen more effectively in citrate-PRP than in PPACK-PRP,indicating that HEP-PG-molecules were most potent in blocking thecation-dependent platelet functions upon collagen stimulation (FIGS. 2,3, 4). It was shown that the heterogeneity of platelet GP Ia/IIaresulted in variability of platelet responses among individuals and thiswas probably reflected in the somewhat variable inhibitory effect ofHEP-PG on the aggregation of the gel-filtered platelets. The resultswere shown to be to be related to the multiple structure of the heparinglycosaminoglycan (HEP-GAG) molecules.

Since, under the conditions used, HEP-PG-molecules appeared not to binddirectly to collagen or to resting platelets, HEP-PG-molecules disturbedplatelet-collagen interaction through other mechanisms. The finding thatalthough HEP-PG did not inhibit Mg²⁺-dependent platelet adhesion, theydid impair the subsequent platelet aggregation (FIGS. 4 and 5), impliedattenuated transmission of the activation signal from GP Ia/IIa to GPIIb/IIIa. Thus, the detected decrease in fibrinogen binding wassecondary to the impairment of collagen-induced platelet activation. Thesuggestion that HEP-PG did not directly interfere with GP IIb/IIIa wassupported further by normal ADP- and epinephrine-induced aggregation andfibrinogen binding in the presence of HEP-PG. Similar results wereobtainable with HEP-GAG molecules of the present invention.

The present findings implied an impairment of activation which followsadhesion of the platelets on collagen (FIGS. 3, 4, 5), leading toinhibited platelet recruitment in flowing blood at both low and highshear rates. Since HEP-PG did not attenuate Mg²+-dependent plateletadhesion, direct inhibition of GP Ia/IIa as the underlying mechanismcould be excluded. HEP-PG-molecules, being macromolecules with a strongnegative charge, which inhibited not only collagen—but alsothrombin-induced aggregation, could have disrupted the outward movementof negatively charged platelet membrane phospholipids during activationwith agonists (Bevers E M, Comfurius P. Zwaal R F A. Blood Rev 1991; 5:146-154). After GP Ia/IIa-mediated adhesion to collagen the subsequentdecrease in platelet function could have been mediated by the reducedligand binding to GP IIb/IIIa. Indeed, HEP-PG decreased fibrinogenbinding to collagen-stimulated platelets and HEP-GAG was shown to do thesame. Under flow conditions, platelet recruitment to collagen dependscrucially on vWf, and its binding to platelet GP Ib and IIb/IIIa, aswell as to collagen. GP IIb/IIIa could be triggered by thrombin to bindvWf, and under conditions when hirudin is used to freeze thrombinsproteolytic actions 60 s after challenging platelets. HEP-PG alsoreduced the binding of vWf to platelets (See Table II below). Insummary, after platelet adhesion to collagen, HEP-PG blocked plateletactivation and also, by binding tightly to vWf, reduced its availabilityto GP IIb(I(Ia. The mast cell-derived HEP-GAG- and HEP-PG-molecules, bybinding to vWf can also result in disturbed interaction of vWf not onlywith GP IIb/IIIa, but also with GP Ib. Thus, ristocetin-induced plateletaggregation was markedly reduced by HEP-PG (See Table I below). At theconcentration of HEP-PG used, the vWf binding to platelets stimulatedwith ristocetin (static conditions) was not affected, although 100-foldexcess of HMWH did inhibit the vWf binding. In blood flowing at highshear rates, in which HEP-PG inhibited platelet-collagen interaction,these macromolecules could certainly have a more potent effect onvWf-dependent platelet activation than in the static binding assays.Indeed, HMWH has previously been reported to severely impairvWf-dependent platelet functions both in vitro and in viva (Sobel M,McNeill P M, Carlson P L, Kermode J C, Adelman B, Conroy R, Marques D: JClin Invest 1991; 87: 1787-1793). However, HEP-PG enhanced, rather thaninhibited the binding of vWf to collagen, which is mediated also byother domains of the vWf molecule than the Al domain, where the GP Iband heparin binding areas are located. The enhanced binding of vWf tocollagen is assumed to have sealed the platelet-activating domains ofcollagen.

Mast cells, one of the sources of the heparin-like molecules of thepresent invention, are prevalent in the adventitial layer of vesselwalls and in the perivascular areas of venules (Galli S J. N Engl J Med1993, 328: 257-265). They are also present in the arterial intima, thesite of atherogenesis and activated mast cells have been found toinfiltrate into the inflammatory shoulder regions of coronary atheromas,the most common site of rupture (Kovanen P T, Kaartinen M, Paavonen T.Circulation 1995; 92: 1084-1088). Upon activation, such as occurs duringinflammation, mast cells degranulate and exocytose an array of potentvasoactive mediators, of which the short-lived leukotrienes andprostaglandins, platelet-activating factor, and histamine are known tostimulate platelets. Histamine releases endothelial von Willebrandfactor (vWf) and P-selectin, two factors that are important adhesivesignals to platelets and leukocytes (Wagner D D. Thromb Haemost 1993;70: 105-110). Furthermore, mast cells secrete glycosaminoglycans, fromwhich the clinically used heparins are derived (Nader H B, Dietrich CP., in Lane D A and Lindahl U (eds): Heparin: Chemical and BiologicalProperties, Clinical Application. Erward Arnold, London. 1989, pp81-96), whereas activated platelets secrete platelet factor 4, aheparin-neutralizing factor, and heparitinase, a heparin-cleavingendoglycosidase. Thus, there appears to be an interplay between thesetwo cell types after their activation, which has been, however, poorlycharacterized so far.

Whether mast cell activation was involved in hemostasis could beevaluated during anaphylaxis and in mastocytosis, two clinicalconditions in which mast cells become excessively activated. Yet inthese conditions, thrombosis was not prevalent despite the release ofplatelet agonists and potent inflammatory mediators which caused changesin vascular endothelium, i.e. downregulation of its nonthrombogenicproperties, induction of adhesive molecules, increased permeability andeven exposure of subendotheiial structures (Wagner D D. Thromb Haemost1993; 70: 105-110).

It therefore seemed likely, that activated mast cells were also able tocounteract their own thrombogenicity. Previously, in addition to theiranticoagulant potential, the clinically useful high-molecular-weightheparin glycosaminoglycans (HMWH; average MW 15 kDa) had been shown toinhibit the platelet aggregation induced by low-dose collagen (FernandezF, N'guyen P. van Ryn J, Ofosu F A, Hirsh J, Buchanan M R. Thromb Res.1986; 43: 491-495); interestingly, this inhibitory effect of heparin wasfound to be directly related to the molecular weight of the heparinsused.

In the studies leading to the present invention, the HEP-GAG- andHEP-PG-molecules were released from mast cell granules after theirexocytosis. The residual proteoglycans that form the insoluble matrix ofthe granules after release of the soluble HEP-PG (the granule remnants;diameter 0.5-1.0 μm) were composed solely of heparin glycosaminoglycanchains (Lindstedt K, Kokkonen J O, Kovanen P T. J Lipid Res 1992; 33:65-74). On the other hand, the soluble proteoglycans released from thegranules into the extracellular fluid contained heparin and to a smallextent also chondroitin sulphate glycosaminoglycan chains (Lindstedt K,Kokkonen J O, Kovanen P T. J Lipid Res 1992; 33:65-74). The differentialeffects of heparinase and chondroitinase treatment on the solubleHEP-PG, the former decreasing their inhibitory activity and the latternot, revealed that the inhibitory effects on platelet-collageninteraction were due to the heparin glycosaminoglycan component of theHEP-PG. In structural analysis of the soluble HEP-PG the composition ofdisaccharide units is typical of heparin (J-p. Li, P. Kovanen, U.Lindahl, unpublished results). Therefore, it could be concluded that theobserved functional differences between HEP-PG and commercial heparinsdepended on other factors than the composition of the glycosaminoglycanchains. Thus, the ability of intact HEP-PG (MW 750 kDa) to inhibitplatelet function was greater than that of the heparin glycosaminoglycanchains (MW 75 kDa) released from HEP-PG, which, again, was greater thanthat of HMWH (MW 15 kDa) or LMWH (MW 5 kDa) (FIG. 2). Taken together,the above findings indicated that both the large size of the heparinchains and their attachment to a core protein to create high molecularweight heparin proteoglycans (HEP-PG) of the present invention were themost important factors contributing to the observed inhibition.

The inhibitory potential of mast cell-derived HEP-PG was related to theheparin chains only, since destroying this moiety with heparinaseeliminated the inhibitory potential. After the HEP-PG was treated withhigh salt concentrations to detach the ion-mediated binding ofproteases, i.e. chymase, tryptase and carboxypeptidase A attached to theglycosaminoglycan chains, the described inhibitory capacity againstcollagen-induced platelet aggregation was present.

Protamine sulphate, known to neutralize the negative charge of thestandard heparin at equimolar concentrations prevented the effect ofHEP-PG only at a 100-fold molar excess. This could indicate thatplatelet-derived factor 4 which is the natural antagonist of heparin andis released during platelet activation, would be needed in extremelyhigh local concentrations to neutralize HEP-PG. HEP-PG retained theinhibitory potential even in the presence of platelet agglutinatingconcentrations of polylysine having the molecular weight of 500 000. Allthis evidence indicated that the strong negative charge of the heparinchains is crucial for the activity of HEP-PG.

Platelets interacting with collagen exposed by vascular injury arebelieved to play a crucial role in both hemostasis and atherothrombosis.The importance of vascular collagen in platelet-vessel wall interactionsis clear in patients with bleeding disorders in whom collagen synthesisis defective, by platelet defects of glycoprotein receptors for collagenor mabs against GP Ia/IIa, and by the enhanced thrombogenicity of smoothmuscle cell matrix when collagen synthesis is optimized. Mastcell-derived HEP-PG could be secreted locally into the subendotheliumand adventitia where mast cells were present and where they could beactivated by various stimuli (Kolodgie F D, Virmani R, Cornhill J F,Herderick E E, Smialek J. J Am Coil Cardiol 1991; 17: 1553-15601. Thesignificant inhibitory capacity of HEP-PG in platelet reactivity towardscollagen, implied a novel mast cell-dependent physiological mechanismlimiting thrombosis in the vascular wall. The mechanism was believed todepend upon the multiple structure of the heparin glycosaminoglycan(HEP-GAG) molecules. The initial findings were later confirmed and couldbe found also in other heparin-like compounds.

Another aim of the present study was to assess the effects of mastcell-derived heparin proteoglycans (HEP-PG) on platelet-collageninteractions. A model with rat serosal mast cells was used. These cellsare filled with cytoplasmic secretory granules composed of HEP-PG with amolecular weight of 750 kDa (range 750 to 1000 kDa), each monomercontaining, on average, ten heparin glycosaminoglycan chains with amolecular weight of 75 kDa (range 50 to 100 kDa) (Yurt R W, Wesley Leid,Jr. R, Austen K F. J Biol Chem 1977; 252: 518-521; Lindstedt K, KokkonenJ O, Kovanen P T. J Lipid Res 1992; 33: 65-74).

Upon activation, mast cells expelled some of their granules into theextracellular fluid where a fraction of the granule HEP-PG becamesolubilized (Lindstedt K, Kokkonen J O, Kovanen P T. J Lipid Res 1992;33: 65-74). It was found that these soluble HEP-PG strongly inhibitedcollagen-induced platelet aggregation and platelet interaction withimmobilized collagen. The findings implied that heparin proteoglycans(HEP-PG) containing multiple heparin glycosaminoglycans (HEP-GAG) orHEP-GAG-molecules as such attenuated the reactivity of platelets to thevascular extracellular matrix, thereby counteracting the other,potentially thrombogenic effects of mast cells. The multiple characterof the HEP-GAG-molecular structure was shown to be very important toachieve the desired effect and in the present invention it has beenshown that the activity of mast cell-derived HEP-GAG-molecules as wellas of other heparin-like compounds is based on a molecular multiplicityand/or high coupling density of negatively charged heparin orheparin-like glycosaminoglycans.

The improvement obtained by the heparin-like compounds of the presentinvention, especially the mast cell-derived HEP-GAG- andHEP-PG-molecules, was shown to be based on the fact, that it completelyinhibits collagen-induced platelet aggregation and serotonin release inplatelet-rich plasma. Furthermore, the heparin-like compounds of thepresent invention should preferably inhibit platelet deposition oncollagen-coated surfaces in flowing whole blood both at low and highshear rates. In the same way as the mast cell-derived multiple HEP-GAG-and HEP-PG molecules, they should preferably attenuate plateletactivation and aggregation as well as fibrinogen binding to plateletsafter collagen stimulation, simultaneously by strongly binding to vonWillebrand factor (vWf) and enhancing its binding to collagen, likelysealing some crucial platelet-activating domains of collagen. Theheparin-like compounds should preferably also reduce thrombin-inducedaggregation and subsequent binding of von Willebrand factor toglycoprotein GPIIb/GPIIIa-complex. Inhibition of collagen-inducedplatelet aggregation in platelet suspension and platelet interactionwith immobilized collagen in flowing whole blood should be their mostprominent property.

The fact that allergen-induced mast cell activation has been shown tosignificantly prolong bleeding time and diminishing thrombin generationin bleeding time blood in volunteers (Kauhanen P. Kovanen P T, ReunalaT, Lassila R. Thromb Haemost, Thromb Haemost 1998; 79:843-7) likelyexplain skin mast cell activation-induced prolongation of bleeding timeby the attenuated reactivity of platelets to vascular extracellularmatrix as a property of mast cell-derived heparin. proteoglycans(HEP-PG) and multiple heparin glycosaminoglycans (HEP-GAG).

Mast cell-derived HEP-PG was shown to abolish thrombus formation on ratfemoral arteries during anastomosis in an acute model lasting 10 mm anda follow-up model of 72 hours—a desired property of the heparin-likecompounds of the, present invention. It was shown that the improvementsobtained especially with HER-GAG- and HEP-PG-molecules are obtainablealso with some other heparin-like compounds of the present inventionhaving the defined technical features and consequently it was believedthat the improvements including platelet aggregation inhibition inconnection with vascular or microvascular injuries and other severedisorders in the vascular system caused by e.g. thrombosis could beobtained with other heparin-like compounds of the present invention. Theheparin-like compounds of the present invention, including the mastcell-derived HER-GAG- and HEP-PG-molecules were shown to offer locallymore efficient prophylactic treatment and prevention of disorders thanunfractionated heparin, in which there was undesired or excessive bloodclotting and e.g. post-operative healing. It was shown that heparin-likecompounds of the present invention like mast cell-derived heparinproteoglycans (HEP-PG) containing multiple heparin glycosaminoglycan(HER-GAG) moieties were responsible for the effect of attenuating thereactivity of platelets to vascular extracellular matrix. It was shownthat it was the multiple structure of the HEP-GAG-moieties of the mastcell-derived HEP-PG-molecules or a high coupling density of negativelycharged glycosaminoglycan units, that provide the heparin-like compoundsof the present invention that provided them with—their uniqueproperties. Thus, it was shown that a spheroidal, bottle-brush-likespatial presentation or configuration is of special importance forobtaining the desired effects and consequently, it could be shown thatsynthetic or semisynthetic HER-GAG-molecules with multiple end-to-end-and/or end-to-side-coupled glycosaminoglycan units as such areespecially advantageous and characterized by the same or improvedproperties as compared to the mast cell-derived HER-GAG- andHEP-PG-molecules.

The other heparin-like compounds of the present invention were producedby coupling glycosaminoglycan units, preferably heparinglycosaminoglycan units end-to-end or end-to-side to form sufficientlylarge glycosaminoglycan molecules, and said multiple straight-chained orbranched glycosaminoglycan molecules could be used as such or conjugatedto a natural, synthetic or semisynthetic core molecule, such as aglobular protein or a polypeptide chain, —preferably a short polypeptidechain and their properties were easy to screen—and test.

The heparin-like compounds of the present invention can, after beingfound active, be used in prophylactic treatment of arterial thrombosisassociated with vascular or microvascular injuries and interventions.

The heparin.-like compounds of the present invention are useful for themanufacturing of preparations for local use as well as for coatingdevices, such as stents, grafts and/or extracorporeal circulationsystems. Said preparations, means and devices can subsequently be usedin prophylactic treatment of arterial thrombosis associated withvascular injuries and interventions as well as for prevention ofinteractions with of flowing whole blood with collagen. Thus, theheparin-like compounds of the present invention are used to manufacturepharmaceutical preparations or medicaments for improved, i.e. moreefficient local treatment to prevent thrombosis, includingatherotrombosis and other hemostatic conditions including vascularinjuries and other changes in vascular endothelium with improvedefficiency.

The heparin-like compounds of the present invention are above all usedin-prophylactic treatment of arterial thrombosis associated withvascular or microvascular injuries and interventions and they arelocally applied or administered as such or in combination with suitable,compatible adjuvants, carriers, etc. or as devices, such as stents,grafts, etc., which have been coated with the heparin-like compounds ofthe present invention.

Different types of medicinal devices, such as stents, their use andtheir coatings have been extensively discussed and are described e.g. inthe following patent publications, the contents of which are herewithincorporated into this specification:

WO 98/22162, ER 832618, U.S. Pat. No. 5,718,862, U.S. Pat. No.5,603,722, U.S. Pat. No. 5,583,213, U.S. Pat. No. 5,571,166, U.S. Pat.No. 5,554,182, U.S. Pat. No. 5,618,298, U.S. Pat. No. 5,342,621, U.S.Pat. No. 5,409,696.

Said devices are also commercially available in a multitude of differentforms suitable for different applications. Such products are e.g.Microstent II™ products from AVE (Arterial Vascular Engineering, SantaRosa, Calif. 95403, USA), NIR™ and NIROYAL™ stents from SciMed BostonScientific Corporation, France. The devices are rigid, semirigid,elastic helixes generally made of plastics, silicons, metals, materialsof hydroxylapatit. A specially preferred material for preparations ofstents is polylactic acid but other substances with the desiredflexible, elastic, semirigid or rigid consistence capable of beingcoated with the HER-GAG- and/or HEP-PG-molecules of the presentinventions are in no way excluded from the scope of the presentinvention.

The invention is also related to preparations with pharmaceutical ormedicinal activity for use in prophylactic treatment and/or preventionof thrombosis, including thrombosis associated with vascular injuries,such as angioplasty, stent and/or graft application including othervascular or microvascular surgery.

The methods and materials used to provide the water-soluble,heparin-like compounds of the present invention as well as preparationsand devices containing them and their use are discussed in more detailin the examples below.

The examples are only illustrative and should not be interpreted aslimiting the scope of the invention. One skilled in the art willimmediately recognise the further possibilities to apply this invention.

EXAMPLES DESCRIBING THE METHODS AND MATERIALS USED Example IA Methods ofObtaining Heparin Proteoglycan (HEP-PG)

Heparin Proteoglycans (HEP-PG) Exocytosed by Stimulated Mast Cells

Mast cells were isolated from rat peritoneal and pleural cavities asdescribed (Yurt R W, Wesley Leid, Jr. R, Austen K F. J Biol Chem 1977;252: 518-521). In a standard assay, 10-13×10⁶ mast cells were incubatedin 1 ml of PBS buffer containing 0.1 mg/ml HSA (Red Cross TransfusionService, Helsinki, Finlandj and 5.6 mM glucose. After preincubation (15mm, 37° C.) the cells were incubated for 15 mm with compound 48/80(Sigma Chemical Go) (5 μglml), a specific mast cell agonist, to inducemast cell degranulation. Control experiments showed that compound 48/80does not induce platelet aggregation. The degranulated mast cells were,then sedimented by centrifugation at 150×g for 10 min, the supernatantwas centrifuged for a further 15 mm at 12 000 g- to sediment theexocytosed granules, and the granule-free supernatant was analyzed forits content of Alcian Blue-reactive (Fluka) material (Lindstedt K,Kokkonen J O, Kovanen P T. J Lipid Res 1992; 33:65-74). In theexperiments performed in the absence of plasma, soybean trypsininhibitor (Sigma) was included to inactivate mast cell-derived neutralserine proteases. Throughout the experiments, HEP-PG were compared withup to 300-fold concentrations (measured as AlcianBlue-reactive-material) of commercial porcine unfractionated,high-molecular-weight heparin (HMWH) (Leiras, Finland) (average MW 15kDa, 1%<7.5 kDa, 1 mg=100 IU USP) and fractionated low-molecular-weightheparin (LMWH) (Fragmin, Kabi Pharmacia) (average MW 5 kDa, 25%>7.5 kDa,1 mg=152 anti Xa units). HEP-PG did not alter the content of ionizedcalcium or magnesium in the buffers or plasma (Microlyte 6, KoneInstruments, Finland) (Boink A B T J, Buckely B M, Christiansen T F,Covington A K, Müller-Plathe D, Sachs Ch, Siggaard-Andersen 0. Eur JClin Chem Gun Biochem 1991; 29: 767-772). HEP-PG were radiolabeled byincubating mast cells with sodium ³⁵S-sulphate (Amersham International),as described (Lindstedt K, Kokkonen J O, Kovanen P T. J Lipid Res 1992;33:65-74). In some experiments HEP-PG were treated with chondroitinaseABC and heparinase (both from Seikagaku Kogyo Go).

Example 1B Methods of Obtaining Heparin Proteoglycan (HEP-PG)

Natural Heparin Proteoglycans (HEP-PG)

Connective-type mast cells, such as skin and serosal mast cells ofmammalian origin can be isolated with the method described in example 1Aor slightly modified methods, not only from rats, but also from othermammalian species such as bovine, swine, sheep, etc. During slaughteringof cows or pigs peritoneal. and pleural lavage is performed withphosphate buffered saline. The pooled fluids are centrifuged once at100×g for 5 mm and the sedimented cells are resuspended in PBS. Theisolation of mast cells is obtained by gradient centrifugation inFicoll, during which mast cells concentrate at the interphase between30% and 40% Ficoll layer. To obtain the soluble proteoglycans mast cellsare stimulated with compound 48/80, a basic polyamine, or calciumionophore A 23187, which induce exocytosis of the mast cell granules.

In contrast to the traditionally isolated bovine or swine-derivedheparin glycosaminoglycans, which are degraded by endoglycosidases,cultured purified mast cells accumulate free, essentially undegradedpolysaccharide chains, which do not undergo endoglycosidic cleavage(Nader H B, Dietrich C R., in Lane D A and Lindahl U (eds): Heparin:Chemical and Biological Properties, Clinical Application. Erward Arnold,London. 1989, pp 81-96). Lymphnode-derived mast cells can be cultured,as well as HMC-1, a human cell line. These cultured cells can begenetically engineered to enhance the production of HEP-PG.Co-cultivation of murine bone marrow-derived mast cells with fibroblastshas been reported to increase biosynthesis of heparin relative tochondroitin sulphate. The use of co-cultivation is recommended in orderto improve proliferation of cells with subsequent release ofHEP-PG-molecules.

Such heparin proteoglycans (HEP-PG) are known to be typically moreresistant to proteolytic degradation, likely due to the high degree ofsubstitution of the peptide core with carbohydrates. This increases thepotential use of the HEP-PG molecules because of their capacity forprolonged activity and due to their increased stability and storabilityor shelf-life.

Example 1 C Methods of Obtaining Human-Derived Natural HeparinProteoglycan (HEP-PG) from Human Mast Cells

Connective-type mast cells, such as skin and serosal mast cells of humanorigin can be isolated with the method described in Example IA orslightly modified methods. It is to be observed that only a small sampleis required, which can be obtained from a patient with routinelyperformed biopsy procedures. The human derived mast cells are thereaftercultivated in a conventional cell culture media and under conditionsallowing good proliferation of the mast cells. The mast cells areharvested with phosphate-buffered saline and treated as described inExample IA and IB. It is also to be observed that once cultured, thecell cultivate can be preserved and stored by per se known methods; andprovides an unlimited source for producing more cells.

Example 1D Methods of Obtaining Heparin Proteoglycan (HEP-PG)

Synthetic High-Molecular Weight Heparin Chains and HEP-PG

The multiple structure of the HER-GAG- and HEP-PG-molecules is essentialfor the inhibitory action upon platelet-collagen interaction. However,also the very long glycosaminoglycan (HEP-GAG) chains, of 75±25 kDa ormore, retain the inhibitory potential, in contrast to the lowermolecular weight species. Thus, the synthetic chains should contain atleast 3-10 heparin glycosaminoglycan units with a molecular weight of atleast 12 but preferably 15-20 kDa, coupled end-to-end or end-to-side toeach other to form straight or branched heparin glycosaminoglycan chainmolecules. To obtain a more native-like construct these separate longheparin (HEP-GAG) chains could also be coupled to a polymeric linkermolecule, as described in the U.S. Pat. No. 5,529,958. Useful polymericcore molecules are for example chain-like peptides comprising repeatedSer-Gly-sequences or globular proteins such as albumin (HSA). which canbe coupled to heparin by the aid of linker molecules such as SPDP,N-succinylimidyl-3-(2-pyridylthio) propionate.

An overdose of heparin to the amount of linker should be used. Butnaturally the proportions can vary depending upon the desired structureof the synthesized heparin-like compound. These both options, i.e.single heparin HEP-GAG-molecules and the synthetic HEP-PG constructsprovide possibilities to prepare improved preparations with lessantigenic properties because the potentially antigenic protein part ofnatural heparin proteoglycans (HEP-PG) is missing. Alternatively to thehigh molecular weight multiple HER-GAG molecules optimal heparindensities can be obtained by coupling standard unfractionated heparin(12 kDa) chains on globular core molecules, such as albumin by means ofSPSD, a heterobifunctional coupling reagent.

Example 1E Heparin Cross-Linking to Bovine Serum Albumin (BSA)

Heparin (Lövens, MW 15 kD) 2 mg was diluted in PBS and coupled toheterobifunctional coupling reagent SPDP(N-succinimidyl-3-(2-pyridylthio)-propionate, Fluka Chemie AG,Switzerland) 1 mg in methanol. The heparin-coupling reagent solution(200 μ1) was activated by OTT (dithiothreitol, Sigma Chemicals Co) 10mg/ml (800 μ1), and the effect of DTT was monitored at 343 nmspectrophotometrically. The sample I ml was eluted through the PD-10column (Pharmacia Biotech, Pharmacia Biotech AB, Uppsala, Sweden) and 10fractions (1 ml each) were collected. The absorbance of the fractionswas analyzed at 343 nm in spectrophotometrically and the four fractionsnot containing free DTT were pooled together.

BSA 0.250 mg (l00 μ1) and SPDP 0.5 mg (50 μ1) were agitated for 20 mm at22° C. and 850 μ1 sodium chloride (0.9%) was added. The sample waseluted through PD-10 column and fractions of 1 ml were collected. Theexclude unbound SPDP 100 μl of the fractions were treated with 900 μ5 ofDTT and absorbance was measured at 343 nm. The five fractions notcontaining free SPDP were pooled. The sodium chloride content was raisedup to 3M in the pooled fractions of both heparin-SPDP and BSA-SPDP, andthese two pools were incubated, together overnight at 4° C. to inducethe coupling. The heparin-BSA coupled sample was eluted with 0.9% NaClthrough Sephacryl S-300 gel (Pharmacia Biotech) (height 30 cm) orthrough PD-10. 0.5 ml fractions 1-45 or 1-25, respectively, werecollected and basing on BSA-content, i.e. absorbance at 280 nm,fractions 24-36 or 16-21, respectively, were collected.

The fractions were concentrated, dialyzed for 30 mm against aqua usingVSWPO2500 film (Millipore Corp. Bedford, Mass.) or minidialyzed(Spectrapor molecular porous membrane MWCO: 12-14.000, Spectrum MedicalIndustries Inc. Laguna Hills, Calif.) for 30 mm and repeatedly for 2hours against aqua. Subsequently, fractions were analyzed forglycosaminoglycan content (Blyscan, Biocolor Ltd. Belfast, NorthIreland) and tested in aggregometer (see FIG. 10). As an example theaggregation curve for PD-10 separated fraction #21 having 0.74 μg/mlheparin is given. All fractions 18-21 showed inhibitory capacity againstcollagen-induced platelet aggregation (for more details see Example 22).

Example 2 Inhibition of Thrombin

The relative potencies of HEP-PG. HMWH, and LMWH were measured withthrombin time in pooled citrated plasma and with a chromogenic assayusing a thrombin substrate (S-2238, Chromogenix, Kabi Pharmacia) (LarsenM L, Abildgaard U, Teien A N, Gjesdal K. Thromb Res 1978; 13: 285-288).In the latter assay, 1 U/ml (110 U/mg) of thrombin (Dade, BaxterHealthcare Co. FL) was the selected dose after titrating the effects ofthe glycosaminoglycan concentrations used. Exogenous thrombin activitywas assessed in the presence of antithrombin III (Kabi Pharmacia) aloneand at two plasma dilutions (1:5 and 1:40 in Tris-NaCl-HSA, pH 8.2) as acontrol for the competitive binding of the glycosaminoglycans to plasmaproteins. In the absence and presence of plasma (1:40 dilution),exogenous antithrombin III was used at two concentrations, 7.5 and 10mU/ml. The reagents were applied to 96-well microtiter plates (Falcon3072, Becton Dickinson) on ice, and incubated for 10 mm at 37° C. S-2238was added, the reaction was stopped with 20% acetic acid, and residualthrombin activity was assessed spectrophotometrically (405 nm)(Labsystems Multiscan MCC, Labsystems, Finland).

Example 3 Platelet Preparation

Blood for the studies was donated by healthy volunteers not using anymedication. Nine volumes of free-flowing blood were collected via a PTFEcannula (Viggo, Sweden) into one volume ofD-phenylalanyl-1-propyl-1-arginine chloromethyl ketone (PPACK)(Calbiochem) (200-400 μM) or acidic citrate dextrose anticoagulant (ACD)[pH 4.9 for aggregation (pH 7.3 in PRP) and pH 4.5 for gel filtration].Platelet-rich plasma (PRP) was separated by centrifugation (180×g, 12mm, 22° C.) and used for platelet aggregation studies and adhesionassays. For detecting deposition of serotonin-positive platelets andrelease reaction, the platelets in PRP were labeled with ¹⁴C-serotonin(specific activity 8 μCi/ml, final concentration of serotonin 40 nM)(Amersham) or 3H-serotonin (specific activity 15 μCi/ml), finalconcentration of serotonin 10 nmol) (Amersham) for 15 mm at 37° C. Inblood perfusion studies, the labeled PRP was added to the remainingblood. The method of platelet detection by serotonin labeling has beenpreviously validated with the determination of deposited protein andwith electron microscopy (Mustonen P. Lassila R. Thromb Haemost 1996;75: 175-181).

Gel-filtered platelets were prepared from PRP after a single washingstep in the presence of PGE₁ (25 ng/ml) and apyrase (1 U/ml) (both fromSigm) and the platelet suspension was then passed through a SepharoseCL-2B column (Pharmacia LKB). After gel filtration, ristocetin (1.0mg/ml) (Sigma) did not induce a platelet response, indicating that vWfwas lacking, and the cell suspension was also devoid of antithrombin IIIactivity, as shown by crossed immunoelectrophoresis (Lane D A, BoisclairM D. in Thompson J M, (ed): Blood Coagulation and Haemostasis. APractical Guide. Churchill-Livingstone. 1991, pp 45-70). Gel-filteredplatelets were used for aggregation studies, for studying Mg²⁺ dependentplatelet adhesion to collagen, and for binding the ligands (fibrinogenand vWf) that mediate platelet-to-platelet interaction. The elutionbuffer was HEPES with 1 mM Mg²⁺ (Timmons S, Hawiger J. in Hawiger J,(ed): Platelets: Receptors, Adhesion, Secretion. Methods in Enzymology.Academic Press, San Diego, Calif. 1989; 169, 11-22). Usually, 2 mM Ca²⁺was added to the suspension of gel-filtered platelets, but when assayingMg-dependent (2 mM) adhesion, Ca²⁺ was omitted (Santoro S A. Cell 1986;46: 913-920). HSA (4%) solution with 2 mM Ca²⁺ and 1 mM Mg²⁺ was usedwhen the platelet-collagen interaction was studied in flowingreconstituted blood without plasma factors (Sakariassen K J, Muggli R,Baumgartner H R. in Hawiger J (ed): Platelets: Receptors, Adhesion,Secretion. Methods in Enzymology. Academic Press, San Diego, Calif.1989; 169: 37-70.) After centrifugations and reconstitutions, the finalplatelet suspension was allowed to stabilize for 30 mm prior to theassays.

Example 4 Platelet Aggregation

Aggregation in PRP and in gel-filtered platelet suspension were studiedwith a Payton aggregometer (Payton Ass. Ltd. Canada). Pepsin-extractedcollagen (Sigma, platelet aggregation kit), and fibrillar type I bovinecollagen (Miller E J, Rhodes R K. Methods in Enzymology. 1982; 82:33-64.), collagen reagent Horm (Nycomed, Hormon Chemie, Germany),thrombin, ristocetin, ADP (Sigma) and epinephrine (Bioanalytical SystemsInc., IN) were used as agonists, each added in a volume of 30 μ1 per 270μl of platelet suspension. The effects of HEP-PG, HMWH, and LMWH werestudied by adding them either during the I-mm preincubation orsimultaneously with the agonist (collagen). In some instances, HEP-PGwere added 10 and 20 s after the collagen. The response was assessed asthe slope of primary aggregation (rate, 1/min) and as maximalaggregation (%).

Example 5 Immobilization of Isolated Fibrillar Collagen

Fibrillar collagen had been extracted from bovine achilles tendon byacetic acid extraction and salt precipitation without pepsin (Miller EJ, Rhodes R K. Methods in Enzymology. 1982; 82: 33-64). Collagen (at aconcentration of 0.36 mg/ml) was kept in 0.5 M acetic acid and fibrilformation induced by neutralizing with 60 mM TES:buffer (1:1) andincubating at 35° C. for 90 mm in humid atmosphere (Holmes D F, CapaldiM J, Chapman J A. Int J Biol Macromol 1986; 8:161-166; Williams B R,Gelman R A, Poppke D C, Piez K A. J Biol Chem 1978; 253: 6578-6585). Foradhesion studies, this fibrillar collagen solution was sprayed fivetimes on ethanol-washed round (diameter 1.5 mm) Thermanox coverslips(NUNC). The successive sprayings of collagen suspension were made justbefore the droplets dried. Collagen settled as a homogeneous layer offibril-containing droplets ranging from 50 to 200 μm with both diametersand interspaces, as assessed by scanning electron microscope (JEQL JSEM820, Japan). The coverslips were kept in a humid atmosphere before useon the same day. For perfusion studies, collagen was immobilized, andnative-type fibrils were allowed to be formed in situ in PTFE tubing(Optinova, Finland) by adding TES and incubating the stoppered tubing at35° C. for 90 mm. After incubation, the tubing was rinsed with PBS.

Example 6 Platelet Interaction with Collagen in PRP or in Mg²+.Buffer

Platelet adhesion to immobilized collagen was studied both in PRP(PPACK) and in gel-filtered platelets in HEPES with 2 mM Mg²+ (Santoro SA. Cell 1986; 46:913-920). Collagen-coated Thermanox coverslips wereplaced on the bottom of the 24-well plates (NUNC) (precoated with 2%HSA) and 1 ml of ¹⁴C-serotonin-labeled PRP or of gel-filtered plateletswith platelet counts adjusted to 100 or to 300×10⁶/ml (Thrombocounter C,Coulter Electronics) was added. Before the assay, the ¹⁴C-scintillationactivity in the platelet suspension and the release of serotonin intoplasma were measured in tubes with imipramine-formaldehyde on ice(centrifuged at 9 500×g for 2 min (Holmsen H, Dangelmaier C A.Measurement of secretion of serotonin, in Hawiger J (ed):

Platelets: Receptors, Adhesion, Secretion. Methods in Enzymology.Academic Press, San Diego, Calif. 1989; 169: 205-210).

After incubation for 30 min either at 22° C. without rotation (to studyadhesion of 100×10⁶ platelets/ml) or at 37° C. during rotation at 100rpm (to study aggregation upon adherent platelets from PRP 300×10⁶platelets/ml), the coverslips were removed, rinsed three times inbuffer, and subjected to scintillation counting. The number of plateletsdeposited on the collagen-coated coverslip was calculated from thenumber of platelets added and from their specific, activity. The releaseof serotonin from the platelets to plasma was also measured asdescribed, and it was constantly below 5%. To assess the role of GPIIb/IIIa under these conditions, PRP was preincubated (15 mm, 37° C.)with a mAb against GP IIb/IIIa (m7E3, kind gift from Dr. Barry Coller)prior to the adhesion assay (Coller B S, Peerschke E I, Scudder L E,Sullivan C A. J Clin Invest 1983; 72: 325-338.

Example 7 Platelet Interaction with Collagen in Flowing Whole Blood orin Reconstituted Blood

To study platelet interaction with collagen in PPACK-anticoagulatedblood (30 ml) containing preincubated ¹⁴C-serotonin-labeled platelets,blood was recirculated for 5 min through the collagen-coated tubing,which was connected to a perfusion pump (Cole Parmer, Ill.). To inducedifferent shear rates (200, 700 and 1700 l/s), at a flow rate of 10ml/mm, tubings of different diameters (1.1, 1.5 and 1.9 mm) were used.The collagen surface was stabilized by perfusing it with PBS (at 37° C.for 15 s) before the blood perfusion. After the perfusion the unattachedplatelets were rinsed off by perfusing with PBS for 30 s. The adherentplatelets were detached by incubating them in 2% SDS twice for 30 minand the lysates were subjected to scintillation counting. In someinstances, scanning electronmicrographs were obtained from the surfaceafter perfusion. Platelet counts, background radioactivity of the blood,and serotonin release were measured, as described for the adhesionassay. To study platelet-collagen interaction in the absence of plasmaproteins, reconstituted blood with washed red cells, buffy coat, andgel-filtered ¹⁴C-serotonin-labeled platelets in HSA solution was used(Sakariassen K J, Muggli R, Baumgartner H R. in Hawiger J (ed):Platelets: Receptors, Adhesion, Secretion. Methods in Enzymology.Academic Press, San Diego, Calif. 1989; 169: 37-70). We also immobilizedstandard heparin (at 10 mg/ml) and HEP-PG (10 μg/ml) to either fibrillarcollagen (Horm) or to monomeric collagen, obtained with pepsin fromnative acetic acid-extracted collagen type I described in the, earlierperfusion experiments, where HEP-PG was used in solution.

Example 8 Interaction Between Platelets and HEP-PG

Binding of HEP-PG to resting platelets was assessed by incubating³⁵S-labeled HEP-PG with PRP or with gel-filtered platelets at 37° C. for15 min. The ³⁵S-scintillation activity was then recovered in plasmafractions and in platelets using sedimentation or gel filtration. HEP-PGwere also immobilized on Thermanox coverslips by incubation for 30 min.The quantity of bound HEP-PG was determined from the ³⁵S-binding, and itwas 56±6 ng/cm² (mean±SD, n=4). Interaction of platelets with HEP-PG wasthen determined using the platelet adhesion assay, as described above.

Example 9 Binding of vWf and Fibrinogen to Activated Platelets

vWf (specific activity 200 U/mg protein) (CRTS, France)(Burnouf-Radosevich M, Burnout T. Vox Sang 1992; 62: 1-11) andfibrinogen were radioiodinated with I¹²⁵ (Amersham) by the method ofBolton & Hunter (Bolton A E, Hunter W M. Biochem J 1973; 133: 529-539).The structural stability of vWf and fibrinogen was confirmed by analysiswith gradient (4-21%) SDS gel electrophoresis. The function ofradiolabeled vWf was confirmed by ristocetin-induced aggregation ofgel-filtered platelets and that of radiolabeled fibrinogen bythrombin-induced coagulation. Gel-filtered platelets were stimulatedwith ristocetin (1 mg/ml) or thrombin (0.1 U/ml) for 3 min. In sometubes thrombin, 60 s after its addition, was inhibited with 3 U/ml ofhirudin. Then ¹²⁵I-vWf (15 μg/ml) was added and the platelets (1×10⁸)incubated at 37° C. without stirring. To separate the platelet-free andplatelet-bound activities, the platelet suspension was layered on top ofa 0.3-M sucrose with 1.35% HSA, and centrifuged at 950×g for 5 min tosediment the platelets. The supernatant was collected, the tip was cutoff, and both fractions were counted for their radioactivity. Thebinding of ¹²⁵I-fibrinogen (100 μg/ml) to AD P-stimulated andcollagen-stimulated (stirred) platelets was studied similarly. The datawere subjected to Scatchard analysis.

Example 10 vWf binding to Collagen and Heparin Proteoglycans (HEP-PG)

The effects of HEP-PG, HMWH, and LMWI-1 on vWf binding to collagen wereassessed according to Lawrie et al. (Lawrie A S, Harrison P, Armstrong AL, Wilbourn B R, Dalton R G, Savidge G F., Br J Haematol 1989; 73:100-104). For this purpose 96-well microtiter wells (Maxisorb, NUNC)were coated for 2 h at 37° C. with pepsinized type I collagen (dialyzedagainst 67 mM phosphate, pH 7.2) (at 50 μg/ml), then washed, and blockedwith 3% BSA. vWf (0.1 pg/ml) was then added to the plates and incubatedfor 2 h in the presence of different concentrations of HEP-PG, HMWH andLMWH. Subsequently, bound vWf was quantified using peroxidase-conjugatedpolyclonal anti-vWf antibody (Dako A/S). In addition, vWf (1 μg) wasincubated with HEP-PG (0.5 μg) for 10 min at 22° C. and applied to acellulose acetate plate (Helena Lab, Tex.). The plate waselectrophoresed for 30 min at 1 80 V in 5 mM HEPES, pH 7.4, containing 2mM Ca²⁺ and 2 mM Mg²⁺, and stained with Alcian Blue to visualize HEP-PGor Ponceau red to visualize vWf.

Example 11 Statistical Analysis

Results are given as mean±SD. The statistical significance of thedifference between sets of values was determined by Student's t-test forpaired values or factorial ANOVA, as indicated.

Example 12 Anastomosis and Two Thrombosis Models of Microsurgery in theRat Femoral Artery

All procedures performed in this study were approved by the appropriateinstitutional guidelines and followed the administrative guidelines foranimal research. Before the procedure the rats were anesthetized(Hypnorm™ 10 ml, Dormicum™ 3 m1, aqua ad 10 ml, i.p). When performingthe anastomosis the femoral artery was exposed, two clamps were set, thevessel was cut, and then flushed with 100 μl of either saline, standardheparin (MW 15 kd, 1 mg/ml, 100 ATU/ml) or HEP-PG (l0 μg/ml). After theprocedure (about 15 min) the vessel was closed by a suture using 8-10stitches with 10-0 microsurgical thread. Circulation was returned for 10min, and the experiment was stopped after this by giving an overdoseanesthesia to the rat. Each treatment mode was tested in 5 rats.

The anastomosis site was excised for scanning electron microscopicevaluation, which was performed without knowing the treatment. Twodifferent magnifications 75 and 500 were taken from each sample. Themicrographs were analyzed by a grading system: 0-3.

In the first thrombosis model (Davidsson S F et al. Plast Rec Surg86:579-582, 1990) the vessel was crushed for 2 min with a bulldog clamp,then the vessel was partly opened, the inner wall was scratched with aneedle for 10 times, then the flushing with either saline, heparin orHEP-PG was performed, and the vessel was closed. Circulation wasreturned for 10 min. The rest of the experiment followed the designprovided in the anastomosis model.

In the other thrombosis model (Andersen D M et al. Microsurgery 15:413-420, 1994) the vessel was treated as in the anatomosis model butwhen closing the suture, the vessel was inverted into the blood streamso that all layers of the vessel, adventitia, media and intima wereexposed to flowing blood. The rest of the experiment followed the designprovided in the anastomosis model.

Examples Showing the Results Example 13 Inhibition of Thrombin

Mast cell-derived soluble HEP-PG at concentrations exceeding 1.0 μg/mlsignificantly prolonged thrombin time when studied in 1:3 plasmadilution (FIG. 1). However, HEP-PG inhibited thrombin significantly lesseffectively than HMWH or LMWH. Also, as measured by the chromogenicassay, HEP-PG were less effective than HMWH in inhibiting thrombin inthe presence of 1:5 plasma dilution. This difference could be observedat the various thrombin concentrations used (0.5-3 U/ml) (data notshown).

To study whether plasma proteins affected the ability of HEP-PG topotentiate exogenous antithrombin III or heparin cofactor II, theeffects of various concentrations of HEP-PG and HMWH on residualthrombin activity were measured in the presence and absence of plasma.At a plasma dilution (1:40), HEP-PG did not differ from HMWH (FIG. 2).In the absence of plasma, however, HEP-PG were more potent than HMWH inenhancing antithrombin III activity. Thus, HEP-PG were able topotentiate antithrombin Ill, but this ability was impaired in thepresence of plasma proteins.

Example 14 Platelet Aggregation and Serotonin Release in Platelet-RichPlasma

Mast cell-derived HEP-PG strongly inhibited collagen-induced plateletaggregation in both types of PRP investigated. When studied incitrate-anticoagulated PRP, HEP-PG were inhibitory at a concentration ofas low as 1.0 μg/ml (FIG. 2). At this concentration, HMWH and LMWH didnot impair aggregation, and these heparins were without effect even if300-fold excess (300 ug/ml) was used. We next treated the HEP-PG withalkali to dissolve their protein components and to obtain isolatedglycosaminoglycan chains. The inhibitory action of the glycosaminoglycanchains, average MW 75 kDa. HEP-GAG was significantly weaker than that ofthe native HEP-PG but significantly better than that of HMWH and LMWH.In contrast to HEP-PG, HMWH impaired collagen-induced aggregation onlyin citrated PRP and at low collagen concentrations (<2.0 μg/ml).

The dose-dependent effects of HEP-PG on collagen-induced aggregation incitrated and in PPACK-anticoagulated PRP showed that the inhibitoryeffect of HEP-PG was independent of collagen concentration up to 150μg/ml, and more pronounced in cation-depleted plasma than in PPACK-PRP,in which total inhibition was reached only at 3 μg/ml. Inhibition wastotal, irrespective of whether HEP-PG and collagen were addedsimultaneously, or HEP-PG were added 10 s after collagen. HEP-PG alsoreduced the release of platelet serotonin from 50% to the backgroundlevel (10%) in PRP, even at the highest collagen concentration tested(150 μg/ml).

In additional experiments we treated HEP-PG with heparinase orchondroitinase ABC. We found that treatment with heparinase totallyabolished the ability of HEP-PG to inhibit collagen-induced plateletaggregation, whereas treatment of HEP-PG with chondroitinase ABC did notlessen their inhibitory potential (not shown). The macroaggregatedHEP-PG complexes forming the granule remnants (i.e., the residues leftover after release of the soluble proteoglycans from the exocytosedgranules (Kovanen P T. Eur Heart J 14 (suppl K) 1992; 105-1 17;Lindstedt K, Kokkonen J O, Kovanen P T. J Lipid Res 1992; 33:65-74) hadno inhibitory effect on collagen-induced platelet aggregation comparedwith the same amount of soluble HEP-PG. However, when the granuleremnants were first disintegrated into HEP-PG monomers by treatment with2 M NaCl and then added to the platelets, the inhibitory effect equaledthat observed with soluble HEP-PG.

The concentration of HEP-PG, which completely abolished thecollagen-induced responses of platelets (3 μg/ml; FIG. 3) was selectedfor testing the effects of HEP-PG on platelet aggregation induced withagonists other than collagen. As shown in Table I, HEP-PG inhibitedplatelet aggregation induced with ristocetin, inhibition being total ata ristocetin concentration of 0.60 mg/ml. Inhibition was alsoconsiderable at the two higher ristocetin concentrations, 0.75 and 1.0mg/mt. HMWH and LMWH did not inhibit ristocetin-induced aggregation tothe same extent as did HEP-PG. Furthermore, HEP-PG did not markedlymodify platelet aggregation in response to ADP or epinephrine (10 μM)(not shown).

Example 15 Aggregation of Gel-Filtered Platelets

When HEP-PG were added to suspensions of gel-filtered platelets, thecollagen-induced platelet aggregation was, only incompletely abolished.With 25 μg/ml of collagen the inhibitory effect of 3 μg/ml of HEP-PGranged between 25 and 60%. We also studied thrombin-induced (0.1 and0.25 IU/ml) aggregation of gel-filtered platelets. Again, plateletaggregation was more effectively inhibited by HEP-PG than by HMWH orLMWH (all at 3.0 μg/ml) (not shown). HMWH, if used at a 100-foldconcentration (300 μg/ml), led to full inhibition at the two thrombinconcentrations used.

Example 16 Interactions Between Platelets and Collagen inMg²+-Containing Buffer and in PRP

In the following, we assessed the interaction of platelets withimmobilized collagen. When 100×10⁶/ml platelets were studied at 22° C.under static, Mg²⁺-dependent conditions, HEP-PG (3 μg/ml) did not affectthe formation of a monolayer of adherent platelets (FIG. 3). Incontrast, HEP-PG significantly inhibited the subsequentplatelet-platelet interaction, when 300×10⁶ lint platelets were rotatedat 37° C. In PPACK-PRP, however, HEP-PG did not significantly decreasethe interaction. Under the corresponding conditions, the mAb against GPIIb/IIIa (m7E3 at 10 μg/ml) inhibited collagen-induced plateletdeposition by 20%, 75%, and 80%, respectively.

Example 17A Efficacy of Soluble HEP-PG on Interactions Between Plateletsand Collagen in Flowing Blood

When PPACK-anticoagulated whole blood was perfused at different shearrates through tubing coated with collagen, HEP-PG (3 μg/ml)significantly inhibited platelet deposition, but not adhesion, on thecollagen. Inhibition was evident both at a low shear rate (200 1/s) andat higher shear rates (700 and 1700 1/s). When the same experiment wasrepeated (at 700 and 1700 1/s) using reconstituted blood without plasma,the platelets adhered to the collagen to the same extent whether HEP-PGwere present or not (not shown). Scanning electron micrographs of theplatelets covering the collagen-coated surface after perfusion withwhole blood at 1700 1/s demonstrated complete absence of aggregates whenHEP-PG were present. At this shear rate platelet adhesion was notsignificantly diminished in the presence of HEP-PG. Surface coverage was22±4% in the absence and 17±5% in the presence of HEP-PG, (n=3).

Example 17B Efficacy of Immobilized HEP-PG to Inhibit PlateletDeposition on Monomeric Collagen-Coated Surface in Flowing Whole Blood

The co-immobilization of collagen with HMWH and HEP-PG did not reducecollagen attachment as evidenced by Blyscan assay (North Ireland,Belfast). When 10 μg/ml of HEP-PG was immobilized on collagen type I(pepsin-treated or isolated from bovine tendon by pepsin) and it wascompared with either buffer or 10 μg/ml of unfractionated heparin HMWH(molecular mass=15 kDa, Heparin, Leiras, Finland), it was found thatHEP-PG reduced platelet recruitment to less than 1 tenth, whereas HMWHdecreased it to one fourth. In addition to being effective in solutionas shown in Example 7A, HEP-PG immobilized upon collagen surface wassimilarly highly effective in blocking platelet-to-platelet interactionin flowing blood. The results are shown in FIG. 6 below.

Example 17C Efficacy of Immobilized HEP-PG to Inhibit PlateletDeposition on Fibrillar Collagen-Coated Surface in Flowing Whole Blood

The co-immobilization of collagen (Horn reagent) with 10 g/ml wascompared with that of 10 μg/ml of unfractionated heparin. In contrast tounfractionated heparin HEP-PG significantly, by 50%, reduced plateletdeposition on the surface. Thus, the efficacy of HEP-PG immobilized withmonomeric collagen could also be obtained over the more native-typecollagen fibers. The results are shown in FIG. 5 below.

Example 18 Binding of Fibrinogen and vWf to Platelets

HEP-PG tended to reduce the binding of fibrinogen to collagen-stimulatedplatelets: from 2.4±1.2 to 1.5±0.7 pmol/10⁸ platelets (n=4, p=0.06), thebackground being 0.8±0.4 pmol/10⁸ platelets, but did not affectADP-induced binding (not shown). However, HEP-PG (3 μg/ml) inhibited vWfbinding to thrombin-stimulated platelets by 40% (234 vs. 349 ng/10⁸platelets (Table II). HWMH at the same concentration was withoutsignificant effect, but at 100-fold excess (300 μg/mi) completelyblocked vWf binding to platelets. HEP-PG did not inhibit vWf binding toristocetin-stimulated platelets. A similar result was obtained withHMWH. Again, 100-fold excess (300 μg/ml) of HWMH significantly inhibitedvWf binding to ristocetin-stimulated platelets.

Example 19 Interaction Between Platelets and Heparin Proteoglycans(HEP-PG)

When HEP-PG were immobilized instead of collagen, and platelets inPPACK-PRP were allowed to attach, the level of platelet deposition was0.36±0.17×10⁶ platelets/cm² (n=4), which did not differ from the valueobtained with immobilized albumin, HSA (0.50±0.31×10⁶ platelets/cm²)(n=4). The finding that platelets did not bind to HEP-PG was confirmedby experiments in which ³⁵S-labeled HEP-PG (3-10 μg/ml) were incubatedin PRP, and after the incubation the platelets were sedimented andcounted for their ³⁵S-scintillation activity. No ³⁵S-scintillationactivity was present in the sediments, indicating that HEP-PG did notcosediment with the platelets. Furthermore, when washed platelets wereincubated with ³⁵S-HEP-PG and subsequently subjected to gel filtration,³⁵S-HEP-PG was eluted separately after the platelet population.

Example 20 vWf Binding to Heparin Proteoglycans (HEP-PG) and to Collagen

vWf and HEP-PG were electrophoresed either alone or together on acellulose acetate plate, and the plates were stained for both proteinand glycosaminoglycans to visualize the individual components. Theaddition of vWf to HEP-PG reversed their mobility from anodic tocathodic, implying an association between vWf and HEP-PG. Since HEP-PGhad inhibited platelet-collagen interaction at a high shear rate andalso interfered with the other vWf-mediated platelet functions (TableI), we studied whether HEP-PG affected vWf binding to collagen, usingELISA assay. vWf binding to collagen was not inhibited, but, on thecontrary, was markedly enhanced. This result differed completely fromthose obtained with HMWH and LMWH. Even at a 10-fold excessconcentration (30 μg/ml), as compared with HEP-PG, HMWH only slightlyincreased the binding of vWf to collagen, and LMWH was without anyeffect.

Example 21 Interaction Between Heparin Proteoglycans (HEP-PG) andCollagen

We also tested the binding of HEP-PG to collagen under conditionsmimicking those in which HEP-PG inhibited the platelet-collageninteraction (i.e., at similar concentrations of HEP-PG and collagen anda similar incubation time). When collagen (whether pepsinized orfibrillar) was immobilized, it did not interact with HEP-PG. Theseresults were obtained using ³⁵S-HEP-PG or detecting glycosaminoglycanswith Alcian Blue. Furthermore, after incubation of collagen with HEP-PG.the pellet obtained by centrifugation through a sucrose cushion failedto show Alcian Blue-reactivity.

Example 22 Collagen Induced Platelet Aggregation in the Presence ofUnfractionated Heparin Cross-Linked with Bovine Serum Albumin (BSA)

Fractions of the unfractionated heparin (12 kDa) cross-linked to bovineserum albumin as described in Example 1E were analyzed in aggregometer(see FIG. 10). As an example the aggregation curve for PD-10 separatedfraction #21 having 0.74 μg/ml heparin is given. All fractions 18-21showed inhibitory capacity against collagen-induced plateletaggregation. Hence, FIG. 10 A illustrates platelet aggregation incitrated PRP preincubated with a buffer 30 μ1. The response depicted isto collagen (Sigma aggregation kit collagen) 30 μ1 at a finalconcentration 25 μg/ml. In FIG. 10 B PRP was preincubated for 1 min with30 μ1 of HEP-PG at the final concentration of 3 μg/ml. No response tocollagen (25 mg/ml) was obtained. In FIG. 10 C PRP was preincubated for1 min with 30 μ1 of fraction #21 albumin-coupled heparin at aconcentration of 0.74 μg/ml (Blyscan). No response to collagen (25μg/ml) was obtained.

Example 23 Results Obtained In Vivo Arterial Thrombosis Models and Modelof Microsurgical Anastomosis

We have obtained in vivo data in rats where HEP-PG is applied topicallyduring femoral artery anastomosis with or without needle-scratch injuryof the surface layers of the vessel and with or without improperplacement of the anastomosis to create blood contact with the deeperlayers of the artery, i.e. intima, media and adventitia. These studiesprovide experimental models for arterial thrombosis during vascularsurgery and microsurgical techniques, which are often hampered bythrombotic complications.

During macroscopic inspection thrombosis occurred in the controlexperiment (saline administration) in nearly all animals operated,patency being 14/22, whereas the 21/22 vessels remained patent whenHEP-PG at 10 μg/ml was administered locally in a volume 100 μ1 for thetime (about 10 min) closing the anastomosis and then allowingnonanticoagulated blood flow over the injury site for 10 min beforesacrificing the rat.

We studied the anastomosis model by infusing 0.5 ml “Indium”-labeled ratplatelets (at 300-500×10⁶/ml) just at the time when releasing blood flowfor ten minutes. Subsequently, the rat was sacrificed, and blood andanastomosis samples were collected. Gamma-counter calculations of thespecific activity of platelets in blood and at the anastomosis sites,revealed significant efficacy for HEP-PG. (111-Indium-labeled platelets:NaCl 14.2±7.2 (n=7) vs. HEP-PG 7.5±2.9, ×10⁶ mean±SD, (n=7)/anastomosisarea, P=0.025, ANOVA).

In scanning electron microscopic analysis significantly less thromboticresponse could be detected in the HEP-PG group in the anastomosis modelwithout any extra provocation of thrombosis (scratching or improperanastomosis). The thrombotic response remained mural and mainlyconsisted of adhesive layer of platelets, and the aggregates were eithersporadic or absent. (FIG. 8). The comparison of scanning electronmicrographs (“blinded” analysis) gave the following scores (min 1, max4): NaCl 3.2 (n=5), HMWH 2.8 (n=5) and HEP-PG 1.8 (n=5), p=0.03, HEP-PGvs. saline in the anastomosis model; ANOVA, repeated measures. Similarresults were obtained, when analysed 72 hours after the anastomosis.

TABLE 1 Effects of glycosaminoglycans on ristocetin-induced plateletaggregation in PRP (critrate). HEP-PGs HMWH HMWH LMWH Ristocetin Control(3 μg/ml) (3 μg/ml) (300 μg/ml) (3 μg/ml) (mg/ml) R MA R MA R MA R MA RMA 0.60 0.7 83 0 <10 0.8 85 0 <10 0.4 81 0.75 6.4 100 0.7 19 5.0 100 1.042 4.7 100 1.00 6.6 100 2.7 59 5.7 100 0.5 52 6.5 100 R = rate ofprimary aggregation (1/min); MA = maximal aggregation (%). Arepresentative platelet aggregation performed on PRP of four donors.

TABLE II Effects of HEP-PG and HMWH on binding of vWf (finalconcentration 5 μg/ml) to stimulated platelets. vWf bound (ng/10⁸platelets) HEP-PG HMWH HMWH Agonist Control (3 μg/ml) (3 μg/ml) (300μg/ml) No agonist 125 ± 8  Thrombin + hirudin 349 ± 72 234 ± 66 312 ±95  108 ± 24 (0.5 U/ml + 3 U/ml) m7E3 + thrombin 138 ± 10 hirudin (10μg/ml + 0.5 U/ml + 3 U/ml) Thrombin, 858 ± 36 808 ± 18 — 188 ± 40 nohirudin (0.5 U/ml) Ristocetin 868 ± 77  960 ± 202 746 ± 174 280 ± 77 (1mg/ml) Values are mean ± SD (n = 4, in duplicate).

Factorial ANOVA: Thrombin+hirudin: HEP-PG vs control, and HEP-PG vs HMWH(300 μg/ml) p<0.0⁵; HMWH (300 μg/ml) vs control, and HMWH (300 μg/ml)vs. HMWH (3 μg/ml) p<0.001. m7E3+thrombin+hirudin: m7E3 vs control,p<0.001. Thrombin, no hirudin: HMWH (300 μg/ml) vs control and vsHEP-PG, both p<0.001. Ristocetin: HMWH (300 μg/ml) vs control, vsHEP-PG, and vs HMWH (3 μg/ml), each p<0.001, —not performed.

The invention claimed is:
 1. Heparin-like compounds consisting ofsoluble heparin-like glycosaminoglycan molecules having a high couplingdensity of negatively charged heparin-like glycosaminoglycan molecules,said molecules comprising several end-to-side connected heparin orheparin-like glycosaminoglycan units, and wherein said molecules inhibitplatelet aggregation upon collagen in flowing blood.
 2. The heparin-likecompounds according to claim 1, wherein the heparin-likeglycosaminoglycan molecules comprise several end-to-end connectedheparin or heparin-like glycosaminoglycan units.
 3. The heparin-likecompounds according to claim 1, wherein the molecular weight of theheparin-like glycosaminoglycan molecules is at least 75±25 kDa.
 4. Theheparin-like compounds according to claim 1, wherein the molecularweight of the heparin-like glycosaminoglycan molecules is more than75±25 kDa.
 5. The heparin-like compounds according to claim 1, whereinthe heparin-like compound is mast cell derived heparin-glycosaminoglycan(HEP-GAG).
 6. Preparations for local administration in connection withvascular or microvascular injuries and/or interventions, characterizedin that they comprise the heparin-like compounds according to claim 1 incombination with pharmaceutically acceptable and compatible carriersand/or adjuvants.
 7. The heparin-like compounds according to claim 1,wherein the heparin-like glycosaminoglycan units comprise commerciallyunfractionated and/or fractionated heparin glycosaminoglycan.